Clinicopathologic report of ocular involvement in ALS patients with C9orf72 mutation

Abstract

Our objective was to present clinicopathologic evidence of anterior visual pathway involvement in patients with amyotrophic lateral sclerosis (ALS) secondary to a C9orf72 mutation. Two related patients from an extended pedigree with ALS and GGGGCC hexanucleotide repeat expansion in the C9orf72 gene (C9-ALS) underwent neuro-ophthalmologic examination. Following death and tissue donation of the younger ALS patient, histopathologic examination of the retina, optic nerve and central nervous system (CNS) was performed. Ophthalmologic examination revealed contrast sensitivity impairment in the younger C9-ALS patient. Immunohistochemistry performed on this patient's donor tissue demonstrated p62-positive, pTDP43-negative perinuclear inclusions in the inner nuclear layer of the retina and CNS. Further colocalization with GLT-1 and recoverin suggested that the majority of retinal p62-positive inclusions are found within cone bipolar cells as well as some amacrine and horizontal cells. In conclusion, this is the first report that identifies disease-specific pathologic inclusions in the anterior visual pathway of a patient with a C9orf72 mutation. Cone bipolar cell involvement within the inner nuclear layer of the retina may explain the observed subtle visual function deficiencies in this patient. Further clinical and histopathologic studies are needed to fully characterize a larger population of C9-ALS patients and explore these findings in other forms of ALS.

Keywords: Pathology; amyotrophic lateral sclerosis; c9orf72; midget bipolar cells; neuropathology; retina; ubiquitin.

Conflict of interest statement

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Figures

Figure 1

Pedigree of C9orf72 family. III-1 underwent complete ophthalmologic clinical exam 12 months prior to their demise, when autopsy was performed. III-5 underwent complete ophthalmologic clinical exam.

Figure 2

C9orf72 (G4C2) repeat expansion detection using repeat-primed PCR. Genomic DNA was amplified using a three-primer system and touchdown PCR cycling program as detailed in the methods and previously described (22,23). In brief, the forward primer contains a 5′ fluorescently labeled tag that hybridizes upstream of the (G4C2) repeat expansion, the reverse primer consists of a non-complementary 5′ tail sequence and three (G4C2) repeat units, and the anchor primer corresponds to the anchor sequence of the reverse primer. As shown in the representative electropherogram, the initial peak corresponds to the shortest fragment containing a minimum of three (G4C2) repeats, with each successive peak corresponding to fragments expanding by one repeat unit. A characteristic saw-tooth pattern is generated as signal intensity decreases as fragment size increases. Electropherograms displaying ≥30 repeats is commonly used as the pathological cut-off value (23).

Figure 3

p62 + immunofluorescence in C9orf72 retina and absence of inclusions in control eye. Abundant p62+ (green) inclusions are seen in the inner nuclear layer of C9-macula (A–C) and peripheral retina (D–F). No p62 + inclusions are seen in control eye (G–I). Sections are counterstained with DAPI (blue). Perinuclear location and crescent morphology of p62 + inclusions in C9orf72 macula are demonstrated on DIC imaging (J, K – inset, arrow). Scale bars = 100 μm.

Figure 4

Colocalization of Poly-(GA)n dipeptide repeat and p62 as well as ubiquitin and p62 in inner nuclear layer of C9orf72 retina. Colocalization of (GA)n dipeptide repeat (red) and p62 (green) is seen in C9orf72 macula INL (A–C). Only background level staining in control macula (D–F). Colocalization of ubiquitin, UB (red) and p62 (green) is seen in C9orf72 macula indicated by the arrow (G–I). In contrast, no colocalization is seen in control macula, only background staining (J–L). Sections were counterstained with DAPI (blue). Scale bars = 100 μm (A–F) and 10 μm (G–L).

Figure 5

Lack of ubiquilin-2 or pTDP-43 immunostaining in p62 + inclusions in C9orf72 macula. No inclusions seen in control macula. No specific ubiquilin-2 (green) staining was seen in C9orf72 (A–C) or control macula (D–F). There was lack of p-TDP-43 (red) colocalization with p62 + inclusions (green) in C9orf72 (G–I). There was no immunostaining in control macula (J–L). Sections were counterstained with DAPI (blue). Scale bars = 100 μm.

Figure 6

Colocalization of p62 (green) and recoverin (Red), in the inner nuclear layer of the macula in a patient with C9orf72 mutation. Figure 6A–C, scale bar = 100 micrometer. Figures 6D–F, correspond to the boxed inset in Figure C, scale bar = 20 micrometers. This figure highlights the fact that around 50% of recoverin + cells (cone midget bipolars) contain intracellular p62 deposits. This figure also illustrates that additional cell types in the INL, not immunostained with recoverin, also harbor these p62 + deposits..

Figure 7

H&E staining of control (A) and C9orf72 (B) macula. No apparent morphologic differences are seen between control and C9orf72 macula. Scale bars = 100 μm.

Figure 8

Control and C9orf72 optic nerve histology. MBP (A,B) and H&E (C,D) staining in control and C9orf72 optic nerve sections. Scale bars = 1 mm (A and B) and 100 μm (C and D).

Figure 9

Lower motor neuron skein-like inclusions and Lewy-like bodies. Phosphorylated TDP-43 positive skein-like inclusions (arrowhead) and Lewy-like body (arrow) in lumbar spinal cord anterior horn neurons. Scale bar = 50 μm.

Figure 10

C9orf72-specific brain pathology. p62-positive small dot-like cytoplasmic inclusions in cerebellar granular neurons (A), ‘star-shaped’ cytoplasmic inclusions in hippocampal pyramidal neurons in (B), and small dot-like cytoplasmic inclusions in hippocampal dentate gyrus (C). Scale bar = 50 μm.