Dorsal root ganglia in Friedreich ataxia: satellite cell proliferation and inflammation

Abstract

Introduction: Dorsal root ganglia (DRG) are highly vulnerable to frataxin deficiency in Friedreich ataxia (FA), an autosomal recessive disease due to pathogenic homozygous guanine-adenine-adenine trinucleotide repeat expansions in intron 1 of the FXN gene (chromosome 9q21.11). An immunohistochemical and immunofluorescence study of DRG in 15 FA cases and 12 controls revealed that FA causes major primary changes in satellite cells and inflammatory destruction of neurons. A panel of antibodies was used to reveal the cytoplasm of satellite cells (glutamine synthetase, S100, metabotropic glutamate receptors 2/3, excitatory amino acid transporter 1, ATP-sensitive inward rectifier potassium channel 10, and cytosolic ferritin), gap junctions (connexin 43), basement membranes (laminin), mitochondria (ATP synthase subunit beta and frataxin), and monocytes (CD68 and IBA1).

Results: Reaction product of the cytoplasmic markers and laminin confirmed proliferation of satellite cells and processes into multiple perineuronal layers and residual nodules. The formation of connexin 43-reactive gap junctions between satellite cells was strongly upregulated. Proliferating satellite cells in FA displayed many more frataxin- and ATP5B-reactive mitochondria than normal. Monocytes entered into the satellite cell layer, appeared to penetrate neuronal plasma membranes, and infiltrated residual nodules. Satellite cells and IBA1-reactive monocytes displayed upregulated ferritin biosynthesis, which was most likely due to leakage of iron from dying neurons.

Conclusions: We conclude that FA differentially affects the key cellular elements of DRG, and postulate that the disease causes loss of bidirectional trophic support between satellite cells and neurons.

Keywords: Dorsal root ganglion; Friedreich ataxia; Gap junction; Inflammation; Monocyte; Satellite cell.

Fig. 1

Immunohistochemistry of glutamine synthetase, S100, and laminin in DRG of normal controls and FA. a-c normal controls; d-f FA; a and d glutamine synthetase; b and e S100; c and f laminin. In normal DRG, reaction product of glutamine synthetase (a) and S100 (b) reveal well organized layers of adjacent and overlapping satellite cells around each neuron. Normal nerve cells are surrounded by delicate layers of laminin (c). In FA, the remaining neurons are smaller, and satellite cell layers are thicker (d and e, insets) and disrupted (d, inset). Laminin reaction product shows multiple layers surrounding neurons, which is consistent with proliferation of satellite cell processes (f, inset). The arrows in (d) and (e) point to residual nodules that are reactive with antibodies to glutamine synthetase (d) and S100 (e), respectively. Bars: (a-f), 100 μm; insets, 20 μm

Fig. 2

Immunohistochemistry and immunofluorescence of the gap junction protein connexin 43 and the satellite cell marker S100 in DRG of normal controls and FA. a-d normal control; e-h FA; a and e positive contrast immunohistochemistry of connexin 43; b and f Alexa 488 immunofluorescence of S100; c and g Cy3 fluorescence of connexin 43; d and h, merged images of b and c, and f and g, respectively. In the normal DRG, connexin 43 shows punctate and coalescing reaction product (a; and inset [arrow]). In FA, a great abundance of connexin 43 reaction product is present in multiple layers around small neurons (e). Hyperplastic satellite cells bridge gaps between neurons. The confocal images of a normal DRG show sparse gap junctions by their punctiform connexin 43 immunofluorescence (c). They are closely related to S100-positive satellite cells (d). In FA, the number of connexin 43-reactive puncta is greatly increased (g and h). Bars: (a) and (e), 20 μm; insets, 10 μm; (b-d) and (f-h), 10 μm. N, neuron

Fig. 3

Immunohistochemistry of mGluR2/3, EAAT1, and Kir4.1 in DRG of normal controls and FA. a-c normal controls; d-f FA; a and d mGluR2/3; b and e EAAT1; c and f Kir4.1. In the normal controls, reaction product displays a single-cell satellite layer around each neuron ([a-c], insets). In FA, neurons are smaller and surrounded by a disorganized layer of multiple immunoreactive satellite cells. Bars: (a-f), 100 μm; insets, 20 μm

Fig. 4

Immunohistochemistry of inflammatory cells in DRG of normal controls and FA. a-b normal controls; c-d FA; a and c CD68; b and d IBA1. a and b show the relative abundance of CD68- and IBA1-immunoreactive cells in normal DRG. Monocytes abut normal neurons (N) ([a and b], insets) but do not penetrate into the neuronal cytoplasm. In FA, a neuron shows invasion by CD68-positive monocytes resembling neuronophagia (inset) (c). Multiple IBA1-positive monocytes lie immediately adjacent to a degenerating nerve cell (N) (d). One ameboid monocyte appears to project thin processes along and through the neuronal plasma membrane (d, inset). Bars: (a-d), 100 μm; (a) and (c), insets, 20 μm; (b) and (d), insets, 10 μm

Fig. 5

Double-label immunofluorescence of IBA1 and S100; and IBA1 and cytosolic ferritin in DRG of normal controls and FA. a-b normal controls; c-d FA. a and c double-label immunofluorescence of S100 (Alexa Fluor 488 green) and IBA1 (Cy3 red); DAPI (blue); b and d double-label immunofluorescence of IBA1 (Alexa Fluor 488 green) and cytosolic ferritin (Cy3 red). In the normal DRG, IBA1-reactive monocytes may gain access to the S100-reactive satellite cell sheath around neurons (N) but remain separated from the neuronal plasma membrane by a thin layer of satellite cell cytoplasm (a, arrow). IBA1 and S100 show no colocalization. In FA, the outline of the S100-positive satellite cell layer appears irregular and disrupted. An IBA1-reactive monocyte (arrow) abuts or penetrates the neuronal plasma membrane (c). The inset in (c) shows infiltration of a residual nodule by IBA1-positive monocytes. DAPI fluorescence in (c) confirms increased cellularity in FA when compared to the normal DRG (a). A normal DRG shows ferritin immunofluorescence in satellite cells and neurons (N) (b). The mixed yellow and green color suggests that ferritin biosynthesis also occurs in IBA1-positive monocytes. In FA, a multilayered rim of satellite cells around a shrunken neuron (N) is intensely fluorescent for cytosolic ferritin (Cy3 red) (d). Ameboid monocytes express both ferritin and IBA1. The inset in (d) shows matching single-color images in further support of the colocalization of IBA1 (Alexa Fluor 488 green) and ferritin (Cy3 red). Bars (all): 20 μm

Fig. 6

Immunohistochemistry of the mitochondrial proteins frataxin and ATP5B in DRG of normal controls and FA. a-b normal controls; c-d FA. a and c frataxin; b and d ATP5B. In a normal control DRG, frataxin reaction product is very prominent only in the neuronal cytoplasm (inset) whereas satellite and other cells in the space between neurons are almost devoid of immunoreactivity (a). In FA, satellite cells express abundant frataxin (c). The inset in (c) shows a neuron (N) that is devoid of frataxin while surrounding satellite cells are immunoreactive (inset, arrows). Similar to frataxin, ATP5B reaction product in mitochondria is prominent in neuronal cytoplasm of normal DRG (b, and inset). In FA, ATP5B reaction product continues to be present in the remaining small neurons but is more prominent in satellite cells (d, arrow, and inset). Bars (a-d), 50 μm; insets, 10 μm