Extensive FUS-immunoreactive pathology in juvenile amyotrophic lateral sclerosis with basophilic inclusions

Abstract

Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a well-recognized entity. However, the molecular underpinnings of this devastating disease are poorly understood. Here, we present genetic and neuropathological characterizations in two young women with fatal rapidly progressive ALS with basophilic inclusions. In one case, a germline mutation (P525L) was detected in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene, whereas no mutation was identified in the other case. Postmortem examination in both cases revealed severe loss of spinal motor neurons with remaining neurons showing basophilic inclusions that contain abnormal aggregates of FUS proteins and disorganized intracellular organelles, including mitochondria and endoplasmic reticulum. In both patients, the FUS-positive inclusions were also detected in neurons in layers IV-V of cerebral cortex and several brainstem nuclei. In contrast, spinal motor neurons in patients with late-onset sporadic ALS showed no evidence of abnormal accumulation of FUS protein. These results underscore the importance of FUS mutations and pathology in rapidly progressive juvenile ALS. Furthermore, our study represents the first detailed characterizations of neuropathological findings in rapidly progressive juvenile ALS patients with a mutation in the FUS/TLS gene.

Figure 1

A schematic diagram of the fused in sarcoma/translocated in liposarcoma protein and the location of the P525L mutation in Case 1. Abbreviations: RRM = RNA recognition motif = RGG = arginine‐glycine rich region; ZnF = zinc finger domain.

Figure 2

Histopathology of juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions in comparison with that of late‐onset sporadic ALS. (A,E) Hematoxylin and eosin (H&E) sections of the spinal cord show the presence of basophilic inclusions in the remaining motor neurons in Cases 1 and 2. (B,F) Immunohistochemical staining shows that the basophilic inclusions are positive for FUS proteins. (C,G) Furthermore, these inclusions are only weakly positive for ubiquitin but negative for TDP‐43 (D,H). (I) In contrast, spinal motor neurons in patients with late‐onset sporadic ALS show no evidence of basophilic inclusions on H&E‐stained section. (J) Furthermore, immunohistochemistry shows the presence of FUS protein in the nucleus of remaining spinal motor neurons. (K,L) Many of the spinal motor neurons in late‐onset sporadic ALS patients show abnormal accumulation of ubiquitinated proteins (K) and TDP‐43 proteins (L). Scale bar in L is 25 µm and applies to all panels.

Figure 3

Ultrastructural analyses of basophilic inclusions in juvenile amyotrophic lateral sclerosis (ALS). (A) A low magnification electron micrograph of the intracytoplasmic inclusion in a spinal motor neuron. The three rectangles in panel A highlight areas of higher magnifications shown in panels B, C and D. (B–D) Higher magnification image at the edge and the center of the inclusion. Both panels B and D show aggregates of disorganized endoplasmic reticulum and mitochondria. (C) In contrast, the center of the inclusion contains filamentous structures that measure 15–20 nm in diameter. (E,F) Immunogold EM using FUS‐specific antibody shows that the majority of FUS proteins are associated with the filamentous structures (E), whereas the immunogold staining for ubiquitin show very few positive gold particles.

Figure 4

Basophilic inclusions can be detected in cerebral cortex and brainstem nuclei in patients with juvenile ALS. (A,B) A hematoxylin and eosin section shows the presence of basophilic inclusion in the cytoplasm of a neuron in the cingulate gyrus (A). Immunohistochemistry using FUS antibody confirms that these inclusions are positive for FUS protein (B). (C–E) The basophilic and FUS‐positive inclusions can also be identified in neurons in the reticular formation in the medulla oblongata (C,D) and the red nucleus (E). (F) In contrast, neurons in the hypoglossal nucleus in both juvenile ALS patients show staining of FUS proteins in the nuclei.

Figure 5

White matter pathology in juvenile amyotrophic lateral sclerosis with FUS‐positive inclusions. (A–C) Hematoxylin‐eosin and Luxol Fast Blue‐PAS (LFB‐PAS) stains of cervical spinal cord show extensive demyelination in the lateral column (LC), but not in posterior column (PC). Arrowheads in panels A and B indicate the motor nerve roots in the anterior horn with severe pallor and loss of myelination. Scale bar in panel C is 100 µm. (D) The density of axons, highlighted by IHC for neurofilament (NF), is slightly reduced in the demyelinated area. Note that many of the axon cylinders show increased diameter (highlighted by arrowheads). Scale bar in panel C is 100 µm. (E,F) In contrast to the lateral column, the myelination in the posterior column in the same patient shows normal density in LFB‐PAS (E), and the axons in the posterior column show uniform diameter (F). The magnifications for panel E and F are the same as in panel C and D, respectively. (G,H) IHC for CD68 and CD3 show extensive infiltration of macrophages and T lymphocytes in the white matter of the spinal cord. Scale bars in panels G and H are 100 µm.