Flagging mitochondria, the powerhouses of the cell, have been linked to general aging and also to a variety of neurodegenerative diseases, including Parkinson and Alzheimer disease (AD). Exactly how mitochondria fit with AD pathology is not yet clear, but an advanced publication in today’s Nature Medicine online offers up an intriguing mechanism. Researchers led by Shi Du Yan at Columbia University, New York, report that amyloid-β (Aβ) binds to the mitochondrial matrix protein cyclophilin D. That protein is a component of the mitochondrial permeability transition pore (mPTP), a potentially lethal channel that leaks damaging reactive oxygen species, calcium, and other harbingers of cellular demise, such as apoptotic cytochrome c, into the cytoplasm. The work suggests that under a torrent of Aβ, mitochondria leakage sets off a cascade that culminates in cell dysfunction or even death. “We think that cyclophilin D will be a very important partner in mediating Aβ-induced mitochondrial dysfunction,” Yan said in an interview with ARF.

The finding “may have implications for the development of mitochondrial targeted therapeutics for AD,” suggested Hemachandra Reddy, Oregon Health Sciences University, Beaverton, in an interview with ARF (and see comment below). In particular, it may help explain the mechanism of action of Dimebon, a drug candidate that is showing promise in early clinical trials (see ARF related ICAD news and ARF related news story). Dimebon has many potential targets in the cell, but one of them is the aforementioned mitochondrial permeability pore, to which it binds with picomolar affinity. “This is actually very nice work and potentially pertinent to Dimebon,” Rachelle Doody, Baylor College of Medicine, Texas, told ARF. “It strengthens our view that mitochondrial permeability pores are somehow central in Alzheimer disease.” Doody is currently overseeing clinical trials of Dimebon, which was previously approved as an antihistamine in Russia.

A growing literature links mitochondria to Alzheimer disease and specifically to Aβ, which has been found lurking in these organelles. Yan’s group previously reported that Aβ interacts directly with the mitochondrial protein ABAD (see Lustbader et al., 2004 and ARF related news story), and just recently researchers in Sweden led by Maria Ankarcrona at the Karolinska Institute, Stockholm, showed that Aβ binds to the mitochondrial transporter TOM, or translocase of the outer membrane, offering up a mechanism for how Aβ gets into the organelles (ARF related news story). Work from several other labs, including Reddy’s, also shows that Aβ attaches to mitochondrial membranes (see, e.g., Manczak et al., 2006). On top of these molecular studies, there is also growing cellular evidence that mitochondria play an intimate role in AD pathology. Cells lacking mitochondrial electron transport chain activity are spared the toxic effects of Aβ, for example (see Cardoso et al., 2001), while “cybrids,” hybrid cells made by replacing mitochondria in normal cells with those taken from AD-damaged ones, show increased Aβ production and deposition (see Khan et al., 2000). “I think it is important for the Alzheimer’s field to note that Aβ is toxic through its effects on mitochondria in general and through its effects on the mitochondrial electron transport chain in particular,” said Russell Swerdlow, University of Kansas, Missouri, in an interview with ARF. “The importance of Yan’s paper is that it provides a potential explanation for those findings,” he said. Swerdlow is a champion of the mitochondrial hypothesis of AD (see Alzforum live discussion).

Using a variety of cellular and transgenic animal approaches, Yan and colleagues show that removing or blocking cyclophilin D protects against Aβ toxicity and attenuates learning deficits in mouse models of the disease. The researchers made the connection by following up their initial studies on ABAD. From immunoprecipitation experiments, first author Heng Du and colleagues found that Aβ binds not only to mitochondrial ABAD but also to cyclophilin D. Subsequently, they showed, using surface plasmon resonance, that the two bind each other and that they co-immunoprecipitate from cortical cell mitochondria taken from AD brain tissue. Confocal microscopy confirmed the colocalization of the two proteins in the cortex of AD brain.

Du and colleagues took advantage of cyclophilin D negative animals and cells to probe the relevance of the Aβ-cyclophilin D connection. The cyclophilin is encoded in the mouse by the Ppif gene, knockouts of which recently became available (see Baines et al., 2005). The researchers crossed the Ppif-/- animals with APP transgenic mice (J20 line) and put both the crosses and the Ppif knockouts through some pertinent tests. Du and colleagues found that mitochondrial calcium uptake, which is reduced in normal aged mice, is further reduced in APP mice but actually enhanced in APP/Ppif-/- crosses. Cyclosporin A, an inhibitor of cyclophilin D, was also able to boost calcium uptake by APP mouse mitochondria, suggesting that cyclophilin D contributes to the age-related decline in calcium buffering capability. Similarly, the researchers showed that knocking out cyclophilin D protects APP mouse cortex against loss of mitochondrial membrane potential, increased production of reactive oxygen species, and diminished respiratory capacity. At the cellular level, the researchers found that while Aβ42 acts on normal cells to decrease mitochondrial potential and increase the release of cytochrome c and apoptosis, the absence of the Ppif gene attenuated these effects. All told, the work suggests that cyclophilin D plays an important role in APP mouse pathology. Exactly how Aβ alters the properties and/or function of cyclophilin D is not yet clear, however. “In an Aβ enriched environment, we believe that more cyclophilin D is translocated to the mitochondrial matrix to increase permeability pore opening,” said Yan.

But would any of these effects contribute to reduced learning and memory, the most obvious symptom of AD? Du and colleagues found that APP/Ppif-/- animals perform significantly better than APP mice in the Morris water maze test of learning and memory. They did not perform as well as wild-type mice, however, suggesting that cyclophilin D is not the sole conduit for pathological events. But in support of the learning and memory experiments, the researchers found that long-term potentiation, a synaptic correlate of memory that is reduced in APP mice, was normal in APP/Ppif-/- animals. Further, they found that Aβ, which inhibits LTP when applied to normal hippocampal slices, had no effect on LTP in Ppif-/- cells. Cyclosporin A was also able to protect normal slices against Aβ-induced LTP deficit.

Turning again to AD itself, the researchers found that cyclophilin D expression is increased in the brain in normal older subjects and is significantly higher yet again in AD patients. “The increased expression of CypD could be an explanation for the observed aging- and Aβ-related impairment of mitochondrial function, as CypD is a key component of the mPTP, and its abundance is associated with the vulnerability of the mPTP to Ca2+,” write the authors.

Whether this age-related cyclophilin D increase is related to the initial trigger that starts the pathological process in sporadic AD is unclear. But that trigger could be related to mitochondria. “In the vast majority of people who do not have autosomal dominant mutations, why in late life do they start making Aβ? My guess is that it is probably something that influences aging and I would say what influences aging is mitochondria going bad,” suggested Swerdlow. If that turns out to be true, then finding ways to protect mitochondria, by, for example, blocking cyclophilin D, could prove to be valuable therapeutically. Whether that is how Dimebon works remains to be determined. “There is now a large preclinical program underway,” said Doody. Medivation, the sponsor of Dimebon, recently announced an agreement with Pfizer to develop the drug (see Medivation press release).—Tom Fagan

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  1. The findings of Du et al. provide an important advance in understanding the mitochondrial amyloid β (Aβ) toxicity in Alzheimer disease (AD) pathogenesis. Their findings suggest that mitochondrial Aβ interacts with the mitochondrial matrix protein, cyclophilin D (CypD), to induce free radical production, increase neuronal oxidative stress, and damage neurons. In addition, they also found that CypD-deficient cortical neuronal mitochondria are resistant to Aβ and Ca2+ induced mitochondrial swelling. Further, CypD-deficient cortical mitochondria have reduced free radical production, and protect neurons from Aβ- and oxidative stress-induced cell death. These important findings further support the mitochondrial oxidative stress hypothesis of AD, and may have some important implications for mitochondrial targeted antioxidant therapeutics in AD.

    Mitochondrial oxidative damage is an early event observed in AD patients and transgenic mouse models of AD (Reddy and Beal, 2008). Further, mitochondrial oxidative damage has been found in peripheral cells (platelets and fibroblasts) from AD patients. Recently, we (Manczak et al., 2006) and others (Crouch et al., 2005; Caspersen et al., 2005; Devi et al., 2006) focused on Aβ and mitochondria and demonstrated the presence of Aβ monomers and oligomers in the mitochondrial membranes. Our digitonin fractionation analysis of isolated mitochondria from APP-transgenic mice revealed Aβ in outer and inner mitochondrial membranes and the mitochondrial matrix. We also showed that mitochondrial Aβ decreases cytochrome oxidase activity, increases free radical production and carbonyl proteins, and damages AD neurons (Manczak et al., 2006). Recently, Hansson Petersen and colleagues reported that Aβ can be transported into mitochondria via the translocase of the mitochondrial outer membrane machinery, and that transported Aβ accumulates on the cristae of mitochondrial inner membrane (Hansson Petersen et al., 2008). These recent discoveries suggest that mitochondrial dysfunction and Aβ play a large role in AD development and progression.

    In the present paper, Du et al. studied the interaction of Aβ and CypD in AD pathogenesis using AD postmortem brains, primary neuronal cultures from CypD knockout mice, APP transgenic mice and double mutant mice (APP transgenic and CypD deficient mice). Using cell biology and electron and confocal microscopy techniques, they found that Aβ interacts with the mitochondrial matrix protein, CypD. To investigate this Aβ interaction with CypD further, these authors crossed CypD knockout mice with APP transgenic mice and studied Aβ pathology, CypD expression, and cognitive behavior in CypD knockout mice, APP transgenic mice, and double mutant mice (APP transgenic and CypD knockout mice). They found that CypD deficiency attenuates Aβ-induced mitochondrial oxidative stress, and improves synaptic function and ameliorates cognitive deficits in double mutant mice. These findings suggest that decreased interaction of Aβ with CypD improves cognitive function in AD.

    However, it is unclear if CypD has a direct role in AD pathogenesis or whether its interaction with soluble Aβ just facilitates the formation of mitochondrial permeability transition pore leading to mitochondrial damage. We need further research to answer these possibilities. It is clear that CypD expression increases with age in APP mice and AD postmortem brains (Reddy et al., unpublished results), and this age-dependent, increased CypD expression may contribute to the opening of mitochondrial permeability transition pore in addition to Aβ interactions with several mitochondrial proteins.

    Findings from this study by Du et al., together with previous studies (Hirai et al., 2001; Swerdlow et al., 1997; Reddy et al., 2004; Lustbader et al., 2004; Manczak et al., 2004; Caspersen et al., 2005; Manczak et al., 2006; Devi et al., 2006; Hansson Petersen et al., 2008), improve our understanding of mitochondrial dysfunction and oxidative damage in AD pathogenesis, and may have implications for the development of mitochondrial targeted therapeutics for AD. Further, recent success of clinical trials of AD patients with Dimebon (which is involved in boosting mitochondrial function) provides additional evidence that mitochondrial targeted therapeutics are promising to improve cognitive function in elderly individuals and patients with AD (Doody et al., 2008; Reddy, 2008).

    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.

    . Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer's disease brain is associated with mitochondrial dysfunction. J Neurosci. 2006 Aug 30;26(35):9057-68. PubMed.

    . Effect of dimebon on cognition, activities of daily living, behaviour, and global function in patients with mild-to-moderate Alzheimer's disease: a randomised, double-blind, placebo-controlled study. Lancet. 2008 Jul 19;372(9634):207-15. PubMed.

    . Mitochondrial abnormalities in Alzheimer's disease. J Neurosci. 2001 May 1;21(9):3017-23. 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.

    . Differential expression of oxidative phosphorylation genes in patients with Alzheimer's disease: implications for early mitochondrial dysfunction and oxidative damage. Neuromolecular Med. 2004;5(2):147-62. PubMed.

    . Mitochondrial medicine for aging and neurodegenerative diseases. Neuromolecular Med. 2008;10(4):291-315. PubMed.

    . Amyloid beta, mitochondrial dysfunction and synaptic damage: implications for cognitive decline in aging and Alzheimer's disease. Trends Mol Med. 2008 Feb;14(2):45-53. PubMed.

    . Gene expression profiles of transcripts in amyloid precursor protein transgenic mice: up-regulation of mitochondrial metabolism and apoptotic genes is an early cellular change in Alzheimer's disease. Hum Mol Genet. 2004 Jun 15;13(12):1225-40. PubMed.

    . Cybrids in Alzheimer's disease: a cellular model of the disease?. Neurology. 1997 Oct;49(4):918-25. PubMed.

  2. The paper is very interesting. Importantly, this study supports our data regarding the localization of Aβ to the mitochondrial inner membrane (Petersen Hansson et al., 2008). They convincingly show that Aβ interacts with cyclophilin D, which is believed to be part of the mitochondrial permeability transition pore. The data suggest a mechanism for how Aβ exerts its toxicity once imported into mitochondria via the TOM import machinery.

    References:

    . The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):13145-50. PubMed.

References

News Citations

  1. Chicago: Dimebon Safe for 18 Months
  2. AD Clinical Pipeline: Immunotherapy Woes, Dimebon Boons
  3. ABAD, aka ERAB: Mitochondrial Miscreant Returns
  4. Aging and Aβ Hit Mitochondria Function

Webinar Citations

  1. A "Mitochondrial Cascade Hypothesis" for Sporadic Alzheimer's Disease

Paper Citations

  1. . ABAD directly links Abeta to mitochondrial toxicity in Alzheimer's disease. Science. 2004 Apr 16;304(5669):448-52. 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.
  3. . Functional mitochondria are required for amyloid beta-mediated neurotoxicity. FASEB J. 2001 Jun;15(8):1439-41. PubMed.
  4. . Alzheimer's disease cybrids replicate beta-amyloid abnormalities through cell death pathways. Ann Neurol. 2000 Aug;48(2):148-55. PubMed.
  5. . Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature. 2005 Mar 31;434(7033):658-62. PubMed.

External Citations

  1. Medivation press release

Further Reading

Primary Papers

  1. . Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease. Nat Med. 2008 Oct;14(10):1097-105. PubMed.