Friedreichs Ataxia Causes Redistribution of Iron

Authors: Arnulf H Koeppen R Liane Ramirez Devin Yu Sarah E Collins Jiang Qian Patrick J Parsons Karl X Yang Zewu Chen Joseph E Mazurkiewicz Paul J Feustel

Publish Date: 2012/05/05

Volume: 11, Issue: 4, Pages: 845-860

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Abstract

Friedreichs ataxia FRDA causes selective atrophy of the large neurons of the dentate nucleus DN High iron Fe concentration and failure to clear the metal from the affected brain tissue are potential risk factors in the progression of the lesion The DN also contains relatively high amounts of copper Cu and zinc Zn but the importance of these metals in FRDA has not been established This report describes nondestructive quantitative Xray fluorescence XRF and mapping of Fe Cu and Zn in polyethylene glycol–dimethylsulfoxide PEG/DMSOembedded DN of 10 FRDA patients and 13 controls Fe fluorescence arose predominantly from the hilar white matter whereas Cu and Zn were present at peak levels in DN gray matter Despite collapse of the DN in FRDA the location of the peak Fe signal did not change In contrast the Cu and Zn regions broadened and overlapped extensively with the Ferich region Maximal metal concentrations did not differ from normal in micrograms per milliliter of solid PEG/DMSO as means ± SD Fe normal 364 ± 117 FRDA 344 ± 159 Cu normal 33 ± 13 FRDA 33 ± 18 and Zn normal 32 ± 16 FRDA 33 ± 19 Tissues were recovered from PEG/DMSO and transferred into paraffin for matching with immunohistochemistry of neuronspecific enolase NSE glutamic acid decarboxylase GAD and ferritin NSE and GAD reaction products confirmed neuronal atrophy and grumose degeneration that coincided with abnormally diffuse Cu and Zn zones Ferritin immunohistochemistry matched Fe XRF maps revealing the most abundant reaction product in oligodendroglia of the DN hilus In FRDA these cells were smaller and more numerous than normal In the atrophic DN gray matter of FRDA antiferritin labeled mostly hypertrophic microglia Immunohistochemistry and immunofluorescence of the Curesponsive proteins CuZnsuperoxide dismutase and Cu++transporting ATPase αpeptide did not detect specific responses to Cu redistribution in FRDA In contrast metallothionein MTpositive processes were more abundant than normal and contributed to the gliosis of the DN The isoforms of MT MT1/2 and brainspecific MT3 displayed only limited colocalization with glial fibrillary acidic protein The results suggest that MT can provide effective protection against endogenous Cu and Zn toxicity in FRDA similar to the neuroprotective sequestration of Fe in holoferritinIn the vast majority of patients with Friedreichs ataxia FRDA the mutation consists of a homozygous guanine–adenine–adenine GAA trinucleotide repeat expansion in the frataxin gene chromosome 9q21 The normal gene product frataxin is critically important for the biogenesis of iron–sulfur clusters though the protein may have several other functions review in ref 1 Among other lesions of the central and peripheral nervous systems FRDA causes selective atrophy of the large neurons of the dentate nucleus DN 2 Smaller nerve cells persist 2 and continue to provide GABAergic afferent terminals to the inferior olivary nuclei In contrast GABAergic corticonuclear connections that normally provide two thirds of synaptic terminals to the DN undergo grumose degeneration and the overall synaptic density declines 3 A recent systematic anatomical study established that loss of large DN neurons constitutes the only critical interruption of the cerebellar module in FRDA 2 and the main correlate to cerebellar ataxia in FRDAThe DN normally contains abundant iron Fe Peculiarly collapse of this gray matter structure in FRDA does not lead to a net decrease or increase of the metal when concentrations are determined on tissue digests 4 The vulnerability of the DN to frataxin deficiency may or may not be related to its high Fe content Several other gray matter structures such as globus pallidus subthalamic nucleus red nucleus and substantia nigra are also Ferich but appear exempt from destruction in FRDA To date there is no direct evidence that Fe in the DN becomes toxic or that the lesion is due to an excess of reactive oxygen species Nevertheless many in vitro models of FRDA show great sensitivity to oxidative stress Bayot et al 5 recently summarized the evidence for the primary role of Fe in the pathogenesis of FRDA and proposed that Fe accumulation in certain systems including the human heart is a secondary event Though Fe may not be critical in the damage to large DN neurons the metal may be important in downstream effects of the primary lesion Koeppen et al 4 documented changes in two Feresponsive proteins of the DN ferritin and ferroportin by immunohistochemistry and analysis of ferritin subunits by Western blotting The results suggested that Fe does not remain inert throughout the course of FRDA 4 implying the presence of ionic iron as a potential catalyst in the Fenton reactionThe DN also contains two other metals with established physiological functions namely copper Cu and zinc Zn The concentrations of these metals are relatively high at 10–15  of total Fe 6 7 8 9 A prior synchrotron study of fixed unembedded slices of human cerebellum confirmed prominent fluorescence of Fe Cu and Zn in the DN but also showed that these metals are only partially colocalized 10 In contrast to Fe the literature contains no information heretofore on the fate of Cu and Zn in the DN of patients with FRDA Inappropriate release of endogenous Cu ions and perhaps Zn ions from DN neurons may contribute to oxidative injury The combined effect of free Fe and Cu is perhaps more serious than that of either metal alone The experimental intracerebral intraventricular or subarachnoid injection of inorganic Cu salts 11 12 is very destructive but no data are available regarding the cells that regulate the metal or the response of specific cuproproteins The experimental intraventricular administration of an organic Cu compound namely Cuhistidine in an effort to explore potential replacement therapy in Menkes disease also causes damage to the exposed brain surfaces 13This report presents qualitative and quantitative observations on the DN in FRDA that were gained through application of nondestructive Xradiation of tissue samples Xray fluorescence XRF of Fe Cu and Zn was correlated with the histopathology of the DN While ferritin is an excellent marker of Fe dysmetabolism similar storage proteins for Cu and Zn do not exist This effort also included the immunohistochemical examination of two cuproproteins that may recognize shifts in brain Cu levels namely CuZnsuperoxide dismutase SOD and Cu++transporting ATPase αpeptide ATP7A Menkes protein and of three metallothionein isoforms

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