Friedreich ataxia: failure of GABA-ergic and glycinergic synaptic transmission in the dentate nucleus

Abstract

Atrophy of large neurons in the dentate nucleus (DN) is an important pathologic correlate of neurologic disability in patients with Friedreich ataxia (FA). Thinning of the DN was quantified in 29 autopsy cases of FA and 2 carriers by measuring the thickness of the gray matter ribbon on stains with anti-glutamic acid decarboxylase, the rate-limiting enzyme in the biosynthesis of γ-amino-butyric acid (GABA). The DN was thinner than normal in all cases of FA, and atrophy correlated inversely with disease duration but not with age at onset or length of the homozygous guanine-adenine-adenine trinucleotide expansions. In 13 of the FA cases, frozen DN tissue was available for assay of frataxin. Dentate nucleus atrophy was more severe when frataxin was very low. Immunohistochemical staining for glutamic acid decarboxylase revealed grumose reaction and preservation of small GABA-ergic neurons in the DN of FA patients. Residual small DN neurons and varicose axons also contained the glycine transporter 2, identifying them as glycinergic. Immunohistochemistry also confirmed severe loss of GABA-A and glycine receptors in the DN with comparable depletion of the receptor-anchoring protein gephyrin. Thus, loss of gephyrin and failure to position GABA-A and glycine receptors correctly may reduce trophic support of large DN neurons and contribute to their atrophy. By contrast, Purkinje cells may escape retrograde atrophy in FA by issuing new axonal sprouts to small surviving DN neurons where they form reparative grumose clusters.

Figure 1

Measurement of dentate nucleus (DN) thickness. Glutamic acid decarboxylase (GAD) stain. (A, B) Ten lines were drawn at right angles to the estimated central axis of the DN. The computer program determined the precise length of lines in µm and stored each measurement. Thinning of the DN and depletion of GAD-reactive terminals in Friedreich ataxia (FA) are evident (A). Analysis of all 3 segments of DN yielded a thickness of 150 ± 26 µm (mean ± SD) for FA13 (A) and 261 ± 22 µm for the normal control (B). Bars, 100 µm.

Figure 2

Correlation of dentate nucleus (DN) thickness and disease duration in Friedreich ataxia (FA). Normal controls are shown by open red triangles, FA carriers by blue circles, and FA cases by open black squares. Progressive thinning of the DN occurs with longer disease durations. Normal DN thickness is 238 ± 21 µm (mean ± SD). The values for both carriers are below the normal range. The linear regression line shown includes only the 29 FA patients and yields a slope of −1.29 at R² = 0.24, p < 0.01.

Figure 3

Immunohistochemistry of non-phosphorylated neurofilament protein and class-III-β-tubulin in the cerebellum of a normal control case and 3 cases of Friedreich ataxia (FA). (A, E) In the normal dentate nucleus (DN), neuron sizes are heterogeneous. Small neurons are generally located at the junction of the DN gray matter and adjacent white matter (arrows). (B–D, F–H) In the FA cases, both immunostains show loss of large neurons in the DN while small neurons remain intact (arrows). Insets show the contrast between morphologically intact Purkinje cells and DN neurons in FA on the same tissue block. A–D, non-phosphorylated neurofilament protein; E–H, class-III-β-tubulin. B, F, patient FA20; C, G, patient FA9; D, H, patient FA29. Bars: 50 µm.

Figure 4

(A–D) Double-label immunofluorescence of class-III-β-tubulin and synaptophysin in Friedreich ataxia (FA) (patient FA27) (A, B) and a normal control (C, D). Class-III-β-tubulin is shown in Alexa 488 (green), synaptophysin in Cy3 (red). The images were acquired by laser scanning confocal microscopy and resolved as maximum intensity projections. (A) A focus of synaptophysin-positive grumose reaction is surrounded and penetrated by a dense network of delicate axons. A neuronal cell body is not distinct in the center of the grumose cluster. (B) Two small neurons ("N") display an abnormally thick layer of synaptic terminals about their cell bodies and proximal dendrites (arrows). This appearance is thought to represent grumose "regeneration" rather than "degeneration". Many thin axons traverse the neuropil. Some are in the immediate vicinity of the synaptic cluster and may be the source of the crowded terminals. (C) Two large normal dentate nucleus (DN) neurons and many nearby dendrites are lined by thin layers of synaptic terminals. Axons in the neuropil are generally thicker than in FA (A, B) and less abundant. (D) A small neuron located in the white matter adjacent to the main DN shows a thin layer of synapses. Axons around the neuron are thicker than in a comparable location of the DN in FA (B). Bars: 20 µm.

Figure 5

(A–H) Immunohistochemistry of glutamic acid decarboxylase (GAD) (A–D) and glycine transporter 2 (GlyT2) (E–H) in the dentate nucleus (DN) in 1 normal control case (A, E) and 3 cases of Friedreich ataxia (FA) (B–D, F–H). GAD reaction product in afferent terminals in the normal DN (A) is very dense and generates negative images of large neurons ("N"). Small GAD-positive neurons are also present. The inset shows a small neuron with GAD-reactive cytoplasm and a layer of GAD-positive synaptic terminals. The cytoplasm of an adjacent large nerve cell ("N") is GAD-negative, but displays a thin layer of axosomatic terminals. In FA patients, GAD immunohistochemistry shows a disorganized DN and several regions of grumose reaction (B, C, arrows). The DN in patient FA29 (D) appears less disorganized though it is thinner than normal and devoid of negative images of neurons and grumose reaction. The inset in (D) shows 2 surviving small γ-amino-butyric acid (GABA)-ergic neurons. The GlyT2 reaction shows punctate product and scattered varicose axons in the normal DN (E). When present, cytoplasmic reaction product occurs only in small nerve cells (E, arrow, and inset). In (F) (patient FA20), punctate GlyT2 reaction product does not appear reduced though the DN is thinner; GlyT2-positive neurons are absent. Reduced GlyT2 reaction product is more convincing in (G) and (H), corresponding to patients FA9 and FA29, respectively. A–D, GAD; E–H, GlyT2. A, E, normal control; B, F, patient FA20; C, G, patient FA9; D, H, patient FA 29. Bars, 50 µm; insets, 20 µm.

Figure 6

(A–L) Immunohistochemistry of γ-amino-butyric acid receptor (GABA)-A-Rγ2, glycine receptor α1/2-subunits (GlyRα1/2), and gephyrin in the dentate nucleus (DN) of 1 normal control case and 3 cases of Friedreich ataxia (FA). In the normal case, GABA-A-Rγ2 (A) and GlyRα1/2 (E), reaction products are most abundant near the plasma membrane of neuronal somata and proximal dendrites, which is more distinctly visible at higher power under oil immersion (insets). In FA cases (B–D, F–H), immunohistochemistry of the receptor proteins generates a peculiar plaque-like distribution of reaction product. Some intact nerve cells also display receptor reaction product (insets). Reaction product is absent from some small neurons (B, F, insets, arrows). Receptor reaction products are more abundant in (D) and (H), respectively, from patient FA29, whose disease began late and continued for 29 years. (I–L) Gephyrin reaction product is present in the cytoplasm of nerve cell bodies and dendrites of the control (I). Immunohistochemistry with anti-gephyrin shows severe loss of large neurons and retention of small nerve cells in FA patients (J–L). In (L) (patient FA29), the neuropil is better preserved than in the cases of early onset (J, patient FA20; K, patient FA9). Some neurons of small or intermediate size are gephyrin-negative (J–L, insets, arrows). A–D, GABA-A-Rγ2; E–H, GlyRα1/2; I–L, gephyrin. A, E, I, normal control; B, F, J, patient FA 20; C, G, K, patient FA9; D, H, L, patient FA29. Bars, 50 µm; insets, 20 µm.

Figure 7

(A–F) Double-label immunofluorescence of γ-amino-butyric acid receptor (GABA-A-Rγ2) and glycine receptor α1/2-subunits (GlyRα1/2) in the dentate nucleus (DN) of a control patient (A–C) and patient FA20 with Friedreich ataxia (FA) (D–F). Images were generated by laser scanning confocal microscopy; fluorescence was obtained by Alexa 488- and Cy3-labeled secondary antibodies. The 2 normal DN neurons (A) (shown as "N") and the single DN neuron in FA 20 (D) ("N") display densely packed GABA- and glycine-receptors in close apposition to each other. Abundance and density of the receptors in the FA neuron do not appreciably differ from the normal state. A, D, GABA-A-Rγ2; B, E, GlyRα1/2; C, F, merged images. Bars, 10 µm.