A mitochondrial matrix protease, which degrades short targeting sequences left over after protein import, can also destroy amyloid-β (Aβ) peptides, according to a paper published online July 18 in the Journal of Biological Chemistry. Elzbieta Glaser and colleagues at Stockholm University and the Karolinska Institute in Stockholm, Sweden, show that the enzyme, presequence protease (PreP, also known as hMP1) cleaves Aβ in vitro and also degrades Aβ added to human brain mitochondrial extracts.

It remains to be seen if PreP regulates Aβ levels in vivo, like the other two Aβ proteases neprilysin and insulin-degrading enzyme (IDE). If this turns out to be the case, then PreP could be an antidote specific to mitochondrial Aβ build-up, which has recently been getting attention for its possible role as an early inducer of oxidative stress in neurons (Caspersen et al., 2005; Manczak et al., 2006).

PreP, which Glaser and colleagues first identified in, of all things, the plant Arabadopsis thaliana, is a zinc metalloprotease which belongs to the same large family of pitrilysins as IDE. PreP functions to clear the mitochondria of the targeting sequences cleaved from proteins that have been imported into mitochondria. Since PreP preferentially degrades short, unstructured peptides, the researchers thought it might also handle Aβ.

Using purified, recombinant human PreP, joint first authors Annelie Falkevall and Nyosha Alikhani showed that the enzyme degraded Aβ1-40, Aβ1-42, and the highly amyloidogenic Aβ artic, producing a unique set of cleavage fragments compared to IDE and neprilysin products. Consistent with its specificity for small, unstructured peptides, PreP did not degrade native insulin, but did chew up isolated insulin β chains. They localized the activity to mitochondria in human brain fractions, and showed that PreP resided in the mitochondrial matrix in rat mitochondria. PreP appeared to be the principal Aβ protease in that fraction based on antibody inhibition experiments.

While the mitochondria matrix may seem an odd location for an Aβ-degrading enzyme, recent studies show that Aβ does accumulate in mitochondria. A high-profile report two years ago suggested that mitochondrial Aβ causes oxidative stress and cell death via inhibition of the Aβ-binding alcohol dehydrogenase (ABAD, see ARF related news story). In the current paper, Glaser and colleagues show that human PreP is completely inhibited under oxidizing conditions, raising the (highly speculative) possibility that Aβ could interfere with its own degradation in mitochondria.—Pat McCaffrey

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  1. This JBC paper by Falkevall and colleagues supports the hypothesis that Aβ enters mitochondria, induces free radicals, and causes oxidative damage early in the progression of Alzheimer disease (Falkevall et al., 2006; Caspersen et al., 2005; Manczak et al., 2006). Findings from this paper are particularly useful in improving our understanding of the degradation of Aβ targeted to mitochondria. In their paper, Falkevall et al. found that mitochondrial peptidasome (or PreP) is localized to the mitochondrial matrix, and is capable of degrading neurotoxic Aβ peptides (1-40 and 1-42).

    Although mitochondrial dysfunction has been described in aging and Alzheimer disease for the last 15-20 years by several groups (including Bruce Ames, Flint Beal, John Blass, Gary Gibson, George Perry, Mark Smith, Russell Swerdlow, Doug Wallace, and others), recent studies by Caspersen et al. (2005), Crouch et al. (2005), Lustbader et al. (2004), and Manczak et al. (2006) have focused on the precise connection between Aβ and mitochondria.

    In support of the hypothesis that Aβ is imported into mitochondria, several recent studies found both monomeric and oligomeric forms of Aβ in mitochondrial membranes (Caspersen et al., 2005; Crouch et al., 2005; Manczak et al., 2006). However, the precise mechanism by which Aβ imports to mitochondrial membranes remains unclear. Caspersen et al. found intracellular Aβ in mitochondria from the brains of patients with AD and in mitochondria from transgenic mice with targeted neuronal overexpression of mutant human APP (Caspersen et al., 2005). They also found a progressive accumulation of Aβ in mitochondrial membranes. Of particular importance is that they detected mitochondria-associated Aβ, principally Aβ1-42, in brain specimens in mice as young as 4 months, before extracellular Aβ deposits developed (Caspersen et al., 2005).

    Using biochemical, immunoblotting, and electron microscopy techniques, our laboratory (Manczak et al., 2006) found a 4 kDa Aβ monomer in isolated mitochondria from the cerebral cortex of Tg2576 mice and also from isolated mitochondria of mouse neuroblastoma cells expressing human mutant APP. Further, using A11 antibody, which recognizes only oligomers (Kayed et al., 2003), we found three distinct bands of oligomers, ranging from 15 kDa to 50 kDa in the mitochondrial pellet from the mutant APP cells, but not in the mitochondrial pellet from the wild-type APP cells, suggesting that both monomers and oligomers of Aβ are associated with mitochondria. In addition, using digitonin fractionation techniques, our laboratory demonstrated that Aβ is primarily localized to the mitoplast (mitochondrial inner membrane plus matrix), and a fraction of Aβ was seen in the outer mitochondrial membrane fraction (Manczak et al., 2006).

    To determine whether mitochondrial Aβ induces free radicals and causes mitochondrial dysfunction, we measured hydrogen peroxide levels, cytochrome oxidase activity, and carbonyl proteins in isolated mitochondria from 2- and 17-month-old Tg2576 mice and age-matched wild-type mice. We found increased levels of hydrogen peroxide in both 2-month-old (25 percent increase with P 0.01) and 17-month-old Tg2576 mice (12 percent increase), compared to the age-matched wild-type mice. We also found increased levels of carbonyl proteins (20 percent increase) and decreased cytochrome oxidase activity (20 percent) in 2-month-old Tg2576 mice, relative to the age-matched wild-type mice. A correlative analysis of Aβ and hydrogen peroxide production in Tg2576 mice revealed that both soluble Aβ1-40 and Aβ1-42 directly correlated with hydrogen peroxide levels but not insoluble Aβ (1-40 and 1-42) (Manczak et al., 2006). These findings clearly suggest that Aβ enters mitochondria, induces free radicals, and causes oxidative damage early in the progression of Alzheimer’s.

    Findings from this study by Falkevall and colleagues, together with previous studies (Lustbader et al., 2004; Caspersen et al., 2005; Manczak et al., 2006), may have implications for the development of mitochondrial therapeutics for Alzheimer disease.

    References:

    . Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease. FASEB J. 2005 Dec;19(14):2040-1. PubMed.

    . Copper-dependent inhibition of human cytochrome c oxidase by a dimeric conformer of amyloid-beta1-42. J Neurosci. 2005 Jan 19;25(3):672-9. PubMed.

    . Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP. J Biol Chem. 2006 Sep 29;281(39):29096-104. PubMed.

    . Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science. 2003 Apr 18;300(5618):486-9. PubMed.

    . ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science. 2004 Apr 16;304(5669):448-52. PubMed.

    . Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet. 2006 May 1;15(9):1437-49. PubMed.

  2. It is surprising and unanticipated that a mitochondrial matrix peptidase would play a role in degrading Aβ. It begs the question of how Aβ, which is normally considered to be a secreted protein, would be mislocalized to the mitochondria matrix.

    View all comments by Charles Glabe
  3. Our group recently discovered that insulin-degrading enzyme (IDE) itself is targeted to mitochondria, as well (Leissring et al., 2004). This mitochondrial isoform of IDE is generated by alternative translation initiation at an upstream codon.

    At the time we published this, it was certainly tempting to speculate that Aβ-degrading proteases present in mitochondria could serve some protective function; however, we did not want to make too much of this absent more convincing evidence that Aβ was actually present in mitochondria. In the intervening years, however, more and more evidence has accumulated to suggest this may very well be the case, as discussed in many of the previous comments above. There are certainly many experiments to do on this interesting topic.

    References:

    . Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria. Biochem J. 2004 Nov 1;383(Pt. 3):439-46. PubMed.

  4. An Intramitochondrial Aβ-cleaving Peptidase

    After identifying human PreP, a mitochondrial zinc-binding metalloendopeptidase that degrades targeting peptides as well as other unstructured peptides within mitochondria, these authors further demonstrated that PreP is able to cleave amyloid peptide (Aβ). Unlike the previously identified mitochondrial ATP-dependent proteases which degrade large, aggregated proteins, PreP uniquely targets peptides up to 70 amino acid residues. Since increasing data have suggested that Aβ accumulates within mitochondria and causes mitochondrial dysfunction, identifying a novel intramitochondrial peptidase capable of degrading Aβ has an important implication in understanding the pathogenesis of Alzheimer disease (AD). We can presume that aging-associated decline of PreP activity due to such causes as oxidative stress, mitochondrial permeability transition, or calcium dysregulation may be an important mechanism leading to impaired “waste removal” within mitochondria in AD, suggesting that protecting or enhancing this enzyme’s activity may be a potential therapy for this devastating disease.

References

News Citations

  1. ABAD, aka ERAB: Mitochondrial Miscreant Returns

Paper Citations

  1. . Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease. FASEB J. 2005 Dec;19(14):2040-1. PubMed.
  2. . Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet. 2006 May 1;15(9):1437-49. PubMed.

Further Reading

Papers

  1. . Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease. FASEB J. 2005 Dec;19(14):2040-1. PubMed.
  2. . Gradual alteration of mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c release. J Bioenerg Biomembr. 2005 Aug;37(4):207-25. PubMed.
  3. . Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet. 2006 May 1;15(9):1437-49. PubMed.
  4. . ABAD enhances Abeta-induced cell stress via mitochondrial dysfunction. FASEB J. 2005 Apr;19(6):597-8. PubMed.

Primary Papers

  1. . Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP. J Biol Chem. 2006 Sep 29;281(39):29096-104. PubMed.