The dorsal root ganglion in Friedreich's ataxia.

Atrophy of dorsal root ganglia (DRG) and thinning of dorsal roots (DR) are hallmarks of Friedreich's ataxia (FRDA). Many previous authors also emphasized the selective vulnerability of larger neurons in DRG and thicker myelinated DR axons. This report is based on a systematic reexamination of DRG, DR and ventral roots (VR) in 19 genetically confirmed cases of FRDA by immunocytochemistry and single- and double-label immunofluorescence with antibodies to specific proteins of myelin, neurons and axons; S-100alpha as a marker of satellite and Schwann cells; laminin; and the iron-responsive proteins ferritin, mitochondrial ferritin, and ferroportin. Confocal images of axons and myelin allowed the quantitative analysis of fiber density and size, and the extent of DR and VR myelination. A novel technology, high-definition X-ray fluorescence (HDXRF) of polyethylene glycol-embedded fixed tissue, was used to "map" iron in DRG. Unfixed frozen tissue of DRG in three cases was available for the chemical assay of total iron. Proliferation of S-100alpha-positive satellite cells accompanied neuronal destruction in DRG of all FRDA cases. Double-label visualization of peripheral nerve myelin protein 22 and phosphorylated neurofilament protein confirmed the known loss of large myelinated DR fibers, but quantitative fiber counts per unit area did not change. The ratio of myelinated to neurofilament-positive fibers in DR rose significantly from 0.55 to 0.66. In VR of FRDA patients, fiber counts and degree of myelination did not differ from normal. Pooled histograms of axonal perimeters disclosed a shift to thinner fibers in DR, but also a modest excess of smaller axons in VR. Schwann cell cytoplasm in DR of FRDA was depleted while laminin reaction product remained prominent. Numerous small axons clustered around fewer Schwann cells. Ferritin in normal DRG localized to satellite cells, and proliferation of these cells in FRDA caused wide rims of reaction product about degenerating nerve cells. Mitochondrial ferritin was not detectable. Ferroportin was present in the cytoplasm of normal satellite cells and neurons, and in large axons of DR and VR. In FRDA, some DRG neurons lost their cytoplasmic ferroportin immunoreactivity, whereas the cytoplasm of satellite cells remained ferroportin positive. Ferroportin in DR axons disappeared in parallel with atrophy of large fibers. HDXRF of DRG detected regional and diffuse increases in iron fluorescence that matched ferritin expression in satellite cells. The observations support the conclusions that satellite cells and DRG neurons are affected by iron dysmetabolism; and that regeneration and inappropriate myelination of small axons in DR are characteristic of the disease.

The pathology of superficial siderosis of the central nervous system.

Chronic or intermittent extravasations of blood into the subarachnoid space, and dissemination of heme by circulating cerebrospinal fluid, are the only established causes of superficial siderosis of the central nervous system (CNS). We studied the autopsy tissues of nine patients by iron histochemistry, immunocytochemistry, single- and double-label immunofluorescence, electron microscopy of ferritin, and high-definition X-ray fluorescence. In one case, frozen brain tissue was available for quantitative assay of total iron and ferritin. Siderotic tissues showed extensive deposits of iron and ferritin, and infiltration of the cerebellar cortex was especially severe. In addition to perivascular collections of hemosiderin-laden macrophages, affected tissues displayed iron-positive anuclear foamy structures in the neuropil that resembled axonal spheroids. They were especially abundant in eighth cranial nerves and spinal cord. Double-label immunofluorescence of the foamy structures showed co-localization of neurofilament protein and ferritin but comparable merged images of myelin-basic protein and ferritin, and ultrastructural visualization of ferritin, did not allow the conclusion that axonopathy was simply due to dilatation and rupture of fibers. Heme-oxygenase-1 (HO-1) immunoreactivity persisted in macrophages of siderotic cerebellar folia. Siderosis caused a large increase in total CNS iron but high-definition X-ray fluorescence of embedded tissue blocks excluded the accumulation of other metals. Holoferritin levels greatly exceeded the degree of iron accumulation. The susceptibility of the cerebellar cortex is likely due to Bergmann glia that serve as conduits for heme; and the abundance of microglia. Both cell types biosynthesize HO-1 and ferritin in response to heme. The eighth cranial nerves are susceptible because they consist of CNS axons, myelin, and neuroglial tissue along their subarachnoid course. The persistence of HO-1 protein implies continuous exposure of CNS to free heme or an excessively sensitive transcriptional response of the HO-1 gene. The conversion of heme iron to hemosiderin probably involves both translational and transcriptional activation of ferritin biosynthesis.

Iron and iron-responsive proteins in the cardiomyopathy of Friedreich's ataxia.

Hypertrophic cardiomyopathy is a common complication of Friedreich's ataxia (FRDA). Histological sections reveal abnormal cardiomyocytes, muscle fiber necrosis, reactive inflammation, and increased endomysial connective tissue. Scattered muscle fibers display perinuclear collections of minute iron-positive granules that lie in rows between myofibrils. Frataxin deficiency in FRDA causes mitochondrial iron dysmetabolism. We studied total iron and the iron-related proteins ferritin, mitochondrial ferritin, divalent metal transporter 1 (DMT1), and ferroportin in FRDA hearts by biochemical and histological techniques. Total iron in the left ventricular wall of FRDA patients (30.7+/-19.3 mg/100 g dry weight) was not significantly higher than normal (31.3+/-24.1 mg/100 g dry weight). Similarly, cytosolic holoferritin levels in FRDA hearts (230+/-172 microg/g wet weight) were not significantly elevated above normal (148+/-86 microg/g wet weight). The iron-positive granules exhibited immunoreactivity for cytosolic ferritin, mitochondrial ferritin, and ferroportin. Electron microscopy showed enhanced electron density of mitochondrial deposits after treatment with bismuth subnitrate supporting ferritin accumulation. The inflammatory cells in the endomysium were reactive for CD68, cytosolic ferritin, and the DMT1 isoform(s) translated from messenger ribonucleic acids containing iron-responsive elements (DMT1+). Progressive cardiomyopathy in FRDA is the likely result of iron-catalyzed mitochondrial damage followed by muscle fiber necrosis and a chronic reactive myocarditis.