A new technique that allows researchers to see the subcellular distribution of iron and other metals in neurons reveals that dopamine-containing neurotransmitter vesicles may function as iron storage depots. The implication is that perturbations in iron-dopamine interactions could contribute to the death of dopaminergic neurons in Parkinson disease, possibly by an increase in oxidative stress.

Consistent with a role for oxidative stress in the disease, another new study shows that the protein products of two different Parkinson-causing genes interact in a single pathway that protects cells against mitochondrial stress, and which appears altered in both sporadic and familial PD.

In the first paper, Richard Ortega and colleagues at Bordeaux University in Gradignan, France, tracked down iron in cells courtesy of a powerful, highly focused X-ray beam emanating from the European Synchrotron Radiation Facility in Grenoble, France. Because of the intense brightness of the beam and the high resolution of the images (down to 90 nm), the researchers were able for the first time to detect iron florescence in subcellular compartments of rat PC12 cells. They found iron mainly in dopamine-containing neurotransmitter vesicles. Overloading cells with iron increased the vesicular signal, particularly in neurite outgrowths and distal ends of the cells. Depleting the cells of dopamine caused a decrease in vesicular iron.

Dysfunctional iron metabolism has been implicated not only in Parkinson disease, but also in other neurodegenerative conditions. Published online in PLoS ONE on September 26, the new method will be generally useful for studying the distribution of iron and other metals in models of these diseases, the authors write. See open-access paper for X-ray fluorescence pictures of iron in dopamine cells.

Iron is essential for normal brain function. Local increases in brain or neuronal iron have been associated with PD, Alzheimer’s, and other neurodegenerative conditions (Oakley et al., 2007; Collingwood et al., 2005). Iron accumulation may even be useful as a biomarker for early PD: an ultrasound signal indicating high iron in the brain region affected by PD has been documented before cells begin to die off. The technique is now being tested as a clinical marker for early PD (Peng et al., 2007). Excess iron could cause increased oxidative damage, a pathway implicated in neurodegeneration generally. However, understanding exactly what iron is doing depends on knowing where it accumulates in cells.

In the new work, Ortega and colleagues first looked at iron distribution in normal dopamine-producing PC12 cells. They observed 200 nm cytosolic structures that contained most of the iron in the cell. Loading cells with additional iron increased the fluorescence associated with the vesicles. The scientists then went on to show that the vesicles also contained dopamine. When the researchers inhibited dopamine synthesis in the cells, the iron content of the vesicles decreased—results that are consistent with an iron-storage function for dopamine. The authors hypothesize that lowered dopamine or faulty storage of the neurotransmitter, both of which have been observed in PD, could raise levels of highly oxidizing cytosolic iron or iron-dopamine complexes, to the detriment of neurons. The researchers saw no such specific localization or changes in potassium or zinc distribution, which they measured with the same technique.

Blaming toxicity on excess iron or highly oxidizing iron-dopamine complexes fits with the idea that death of neurons in PD stems from oxidative stress. In further support of that notion, new work from the labs of Miguel Martins of the MRC in Leicester, UK, and Julian Downward at the Cancer Research UK London Research Institute, demonstrates that the protein product of PINK1, a putative mitochondrial kinase encoded at the PARK6 locus, associates with the apoptotic protease HtrA2 (encoded by the PARK13 gene). PINK1 appears necessary for the phosphorylation of HtrA2 after stimulation of the stress-activated p38 map kinase pathway, and loss of function of either protein sensitizes cells to oxidative stress. Thus, HtrA2, a protein previously proposed to be proapoptotic, appears to help protect cells from mitochondrial stress.

The work, published in the September 30 online issue of Nature Cell Biology, suggests that both proteins contribute resistance to apoptosis in response to mitochondrial stress. Thus, the protective effect recently discovered for PINK1 (Pridgeon et al., 2007 and see review by Abeliovich, 2007) may come at least in part from its ability to modulate HtrA2 activity.

PINK1 mutations cause rare, early-onset Parkinson disease, but the PINK1/HtrA2 pathway could play a role in the more frequent, sporadic form of PD as well, the authors claim. Looking at patient tissue, the investigators found that the phosphorylation of HtrA2 is decreased in patients with early-onset PD due to PINK1 mutations. However, the opposite occurs in sporadic PD, where HtrA2 phosphorylation is increased. Since phosphorylation appears to make HtrA2 more active, the results raise the possibility that HtrA2 is activated as a compensatory response to ongoing stress in sporadic PD, and that further understanding of this pathway could yield insights and novel therapeutic approaches to the most common form of the disease.—Pat McCaffrey.

References:
Ortega R, Cloetens P, Deves G, Carmona A, Bohic S. Iron Storage within Dopamine Neurovesicles Revealed by Chemical Nano-Imaging. PLoS ONE. 2007 Sep 26;2(9):e925. Abstract

Plun-Favreau H, Klupsch K, Moisoi N, Gandhi S, Kjaer S, Frith D, Harvey K, Deas E, Harvey RJ, McDonald N, Wood NW, Martins LM, Downward J. The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nat Cell Biol. 2007 Sep 30; [Epub ahead of print] Abstract

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  1. This is significant in that it provides a new analytical tool for spatially assessing the presence of iron in cells including neurons. Although iron has been reported to be increased in the substantia nigra, the brain region impacted in the disorder, information on cell-specific and/or subcellular localization has been lacking. That is where this group’s finding that iron is present in dopaminergic neurons is most significant for Parkinson disease. When dopamine synthesis was reduced, this resulted in release of iron-dopamine complexes from neurovesicles into the cytoplasm. Release of dopamine from the vesicles could result in increased oxidation, leading to enhanced cell death. This study was performed in the dopaminergic cell line PC12 neuronally differentiated via nerve growth factor. I am not familiar enough with the technique to assess its possible clinical applications (it appears it might only be useful for analyses of postmortem samples), but it does appear as if it would be useful in terms of assessing subcellular iron localization at least in animal models of the disease.

    View all comments by Julie Andersen
  2. Comment by Joanna Collingwood, Jon Dobson, Mark Davidson, Chris Batich, Albina Mikhaylova, and Jeff Terry
    This interesting study by Ortega et al. uses a high-resolution synchrotron X-ray probe in a newly developed configuration at the European Synchrotron Radiation Facility (ESRF). The technique they describe has, in recent years, been embraced by researchers from the medical community. It is good to see facility expansion and development in this area as synchrotron X-ray chemical imaging is adopted as a valuable technique to study biological samples (e.g., Mikhailova et al., 2000; Mikhaylova et al., 2005; Wang et al., 2005; Collingwood et al., 2005; Palmer et al., 2006; McRae et al., 2006; Chwiej et al., 2007; Arora et al., 2007).

    Ortega et al. have achieved an excellent in-plane resolution of 90 nm to map iron, zinc, and potassium. Although subcellular chemical mapping studies are conducted at below 50 nm at facilities such as the Advanced Photon Source (APS) 2-ID beamline, mapping in the hard energy range used by Ortega et al. has usually been performed at pixel resolutions of 150 nm and above (Kemner et al., 2004; Palmer et al., 2006; Finney et al., 2007). The X-ray subcellular imaging technique itself is an established tool in the synchrotron community (Paunesku et al., 2006), and development of a multilayer Laue lens promises resolutions of just a few nanometers in the hard energy X-ray range (Kang et al., 2006).

    Synchrotron X-rays are increasingly being used to map metal ion distributions in human tissues (e.g., Collingwood et al., 2005; Chwiej et al., 2007; Miller et al., 2006) and cell cultures (e.g., Palmer et al., 2006; Ortega et al., 2007). Given that these studies require access to the high-flux, high-energy focused X-ray beams available at third-generation synchrotron facilities such as the APS (Chicago, USA), ESRF (Grenoble, France), and DIAMOND (Oxford, UK), one might reasonably ask why researchers are dedicating such effort towards these studies. With access to a modestly equipped histology lab, it is possible to obtain beautifully stained tissue sections revealing the distribution of specific metals (e.g., iron) and of associated proteins (e.g., ferritin) with relatively little trouble. Likewise, accurate quantification of metal ion concentrations in tissue can be undertaken with readily available techniques such as ICP-MS. Ortega’s latest paper (Ortega et al., 2007) provides a good illustration of some of the benefits the synchrotron approach can bring when compared to more conventional methods.

    If we compare a conventional iron stain with a synchrotron X-ray fluorescence map of iron, there are several significant differences. The staining process cannot reveal iron in all its bound forms, so the spatial distribution of iron concentration may be better revealed by a synchrotron map. The chemical element detection sensitivity available at third-generation synchrotrons with focused X-ray beams is outstanding (a few ppm). There is great flexibility in selecting mapping resolution (from tens of nanometers to hundreds of microns). Moreover, the synchrotron techniques are ideal for imaging iron in unstained, unfixed sections, avoiding the interference/alteration of the iron’s chemical state and distribution in the tissue frequently brought about by standard processing and staining techniques (Dobson et al., 1996; Mikhaylova et al., 2004). This approach enables retrospective staining and/or imaging, allowing image correlation with structures of interest (Mikhaylova et al., 2004). The synchrotron approach also routinely allows for the simultaneous collection of metal maps for a wide range of chemical elements (e.g., Collingwood et al., 2005a; Miller et al., 2006).

    Ortega et al. map the intracellular Fe, Zn, and K concentrations in cultured PC12 rat neurons, and extend the investigation to explore how iron concentration and distribution in isolated neurons is affected by exposure to iron (with FeSO4) and by inhibition of dopamine synthesis (with AMT). This is a good example of how synchrotron radiation can contribute to multi-modal imaging approaches, where multi-metal maps of unstained cells or tissue sections are correlated with light microscopy of the structures, and—in this case—with dopamine fluorescence, allowing colocalization of iron and dopamine to be observed.

    The subject matter of this study is of high importance. While iron accumulation has been quantified in individual unstained unfixed dopaminergic neurons (Oakley et al., 2007), and alterations in regional brain iron are observed in a host of neurodegenerative disorders, including Alzheimer disease, the precise mechanisms by which iron is involved in neurodegeneration are not well understood. The field has gained recent momentum as the application of evolving techniques provides insight into colocalization of metals, regional distributions at cellular and subcellular resolution, and altered chemical and mineral states—all of which are important if we are to understand the contribution of metals to oxidative stress in neurodegeneration.

    One particular advantage to synchrotron studies of iron in cells and neurodegenerative tissues is that the mapping can be combined with absorption spectroscopy containing information about the chemical state of the element (Collingwood et al., 2005; 2005a; 2006; Chwiej et al., 2007). In a single experiment, it is possible to use the X-ray beam to map iron concentrations in tissue, and to then look at the absorption profile of individual localized iron accumulations to determine their form (including oxidation state and local chemical environment—e.g., ferrihydrite from ferritin, or deposits of hemosiderin). An application of this combined mapping and characterization technique is to look in situ at altered forms of iron storage in neurodegenerative disease (Collingwood et al., 2005; Mikhaylova et al., 2005; Chwiej et al., 2007)—an important factor in exploring the underlying chemistry in diseases such as Alzheimer’s and Parkinson’s.

    The excellent sensitivity of synchrotron X-rays to metal ions, whilst being one of the fluorescence mapping technique’s greatest advantages, also necessitates careful preparation of samples. Conventional fixation and staining techniques can alter valence stages and lead to leaching of iron, so ideally tissue should be fresh frozen and sectioned with a non-metallic blade to prevent particulate contamination. Choice of mounting substrate is also important. Many glass and plastic slides contain trace metal levels that are irrelevant to routine microscopy work, but that will be evident in micro- or nano-focus synchrotron fluorescence mapping (Mikhaylova et al., 2004).

    While the synchrotron fluorescence technique is excellent for obtaining relative concentrations over an area of uniform sample thickness, it is more challenging to attempt absolute quantification of elemental concentrations. This is primarily because although the pixel area is defined, it is very hard to define the actual volume sampled by the X-ray beam. For the purpose of interpreting the chemical element maps in a study such as Ortega 2007, it must be recognized that the variation in sample thickness across an isolated cell, even within the area of the beam spot, will affect measured concentrations. The signal intensity is proportional to the absolute number of atoms within the excited volume. Since the X-rays pass through the samples essentially unimpeded, the entire thickness is excited. If the thickness of the sample varies in an unmeasured way, as for example, it does across the isolated cells in these samples, then the actual concentration of the metal cannot be determined.

    Another challenge, particularly when working with intact tissue sections, is location of nanoscale regions in unstained sections to enable retrospective image correlation. Following on from our initial use of zinc wire grids (Mikhailova et al., 2000; Mikhaylova et al., 2005) in avian tissue studies, we have proposed the use of numbered lithographic grids deposited onto the slides to facilitate co-registration of images (Collingwood et al., 2005a) and in fact have used lithographically formed un-numbered grids in studies in 2000 and 2001. Where iron is the primary metal of interest, chrome is an ideal choice for the grid, providing a metal signal in the vicinity of, but not overlapping with, the iron. For samples with clearly identifiable metal-bearing features that can be collocated with visual landmarks such as tissue section edges, fissures, or iron rich blood vessels, for example, such grids are unnecessary.

    It should be noted that other methods are also available to obtain nano-resolution multi metal maps of cells and tissues. This is illustrated in Quintana 2007, where detailed high-resolution (50-100 nm) studies of metals and their relation to cellular and subcellular structures have been performed on Alzheimer’s tissue using nano secondary iron mass spectrometry (nanoSIMS). One of the significant differences between this technique and the synchrotron X-ray approach is that the latter is non-destructive and the nanoSIMS technique cannot yield chemical state information. There is also some issue with the SIMS technique with matrix effects that can cause very large shifts in sensitivity with slight changes in composition of the matrix.

    The synchrotron experiments discussed here are not appropriate for clinical imaging, being directed toward autopsy tissue and in-vitro experiments. Synchrotron radiation has been used for non-invasive coronary imaging since 1987 (Rubenstein et al., 1987; Bertrand et al., 2005), but it is not currently viable for high-resolution mapping of trace metals. Amongst many practical difficulties, perhaps the primary one is the multi-hour imaging times and unacceptable resulting dose to which a patient would be exposed.

    However, altered regional brain iron accumulations hold potential for non-invasive clinical detection and diagnosis in neurodegenerative disorders. In particular, the impact of iron on T2 shortening in MRI is being investigated for diseases including Alzheimer’s and Parkinson’s disease (House et al., 2007; Bartzokis et al., 2004; Schenk et al., 2006; Graham et al., 2000).

    Meanwhile, it is important that metal-related mechanisms of neurodegeneration continue to be investigated, particularly where results may influence therapies such as metal chelation (Youdim et al., 2004). Synchrotron X-ray fluorescence imaging is a standard tool for scientists in a variety of disciplines, but its recognition as a tool to study the role of metals in neurodegenerative tissues is comparatively recent. There is great potential for creative synchrotron experiments to enable progress in this fascinating area of neuroscience.

    See also:

    Barrea et al., The BioCAT Microprobe for X-ray Fluorescence Imaging, MicroXAFS and Microdiffraction Studies on Biological Samples, Proc. 8th Int. Conf. X-ray Microscopy IPAP Conf. Series 2006, 7 230-232.

    Collingwood et al. High-resolution x-ray absorption spectroscopy studies of metal compounds in neurodegenerative brain tissue, J. Phys. Conf. Ser. 2005a, 17 54–60.

    Mikhailova et al., Mapping and Characterization of Iron-Containing Particles in Brain Tissue, APS Activity Report 2000.

    Mikhaylova et al., Iron Biominerization of Brain Tissue and Neurodegenerative Disorders, Ph.D. Dissertation, University of Florida, 2004.

    View all comments by Joanna Collingwood

References

Paper Citations

  1. . Individual dopaminergic neurons show raised iron levels in Parkinson disease. Neurology. 2007 May 22;68(21):1820-5. PubMed.
  2. . In situ characterization and mapping of iron compounds in Alzheimer's disease tissue. J Alzheimers Dis. 2005 Aug;7(4):267-72. PubMed.
  3. . Iron and paraquat as synergistic environmental risk factors in sporadic Parkinson's disease accelerate age-related neurodegeneration. J Neurosci. 2007 Jun 27;27(26):6914-22. PubMed.
  4. . PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1. PLoS Biol. 2007 Jul;5(7):e172. PubMed.
  5. . Parkinson's disease: pro-survival effects of PINK1. Nature. 2007 Aug 16;448(7155):759-60. PubMed.
  6. . Iron storage within dopamine neurovesicles revealed by chemical nano-imaging. PLoS One. 2007;2(9):e925. PubMed.
  7. . The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nat Cell Biol. 2007 Nov;9(11):1243-52. PubMed.

External Citations

  1. open-access paper

Further Reading

Papers

  1. . Iron dysregulation and neurodegeneration: the molecular connection. Mol Interv. 2006 Apr;6(2):89-97. PubMed.
  2. . Iron storage within dopamine neurovesicles revealed by chemical nano-imaging. PLoS One. 2007;2(9):e925. PubMed.
  3. . The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nat Cell Biol. 2007 Nov;9(11):1243-52. PubMed.
  4. . The modulation of metal bio-availability as a therapeutic strategy for the treatment of Alzheimer's disease. FEBS J. 2007 Aug;274(15):3775-83. PubMed.
  5. . Iron and paraquat as synergistic environmental risk factors in sporadic Parkinson's disease accelerate age-related neurodegeneration. J Neurosci. 2007 Jun 27;27(26):6914-22. PubMed.
  6. . Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease. Neuron. 2003 Mar 27;37(6):899-909. PubMed.

Primary Papers

  1. . Iron storage within dopamine neurovesicles revealed by chemical nano-imaging. PLoS One. 2007;2(9):e925. PubMed.
  2. . The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1. Nat Cell Biol. 2007 Nov;9(11):1243-52. PubMed.