Impaired Nucleoporins Are Present in Sporadic Amyotrophic Lateral Sclerosis Motor Neurons that Exhibit Mislocalization of the 43-kDa TAR DNA-Binding Protein

Abstract

Background and purpose: Disruption of nucleoporins has been reported in the motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS). However, the precise changes in the morphology of nucleoporins associated with the pathology of the 43-kDa TAR DNA-binding protein (TDP-43) in the disease process remain unknown. We investigated the expression of nucleoporins that constitute the nuclear pore complex (NPC) in spinal motor neurons that exhibit sALS in relation to TDP-43 pathology, which is a reliable neuropathological hallmark of sALS.

Methods: Paraffin-embedded sections of the lumbar spinal cord were obtained for immunofluorescence analysis from seven control subjects and six sALS patients. Anti-TDP-43 antibody, anti-nucleoporin p62 (NUP62) antibody, and anti-karyopherin beta 1 (KPNB1) antibody were applied as primary antibodies, and then visualized using appropriate secondary antibodies. The sections were then examined under a fluorescence microscope.

Results: NUP62 and KPNB1 immunoreactivity appeared as a smooth round rim bordering the nuclear margin in normal spinal motor neurons that exhibited nuclear TDP-43 immunoreactivity. sALS spinal motor neurons with apparent TDP-43 mislocalization demonstrated irregular, disrupted nuclear staining for NUP62 or KPNB1. Some atrophic sALS spinal motor neurons with TDP-43 mislocalization presented no NUP62 immunoreactivity.

Conclusions: Our findings suggest a close relationship between NPC alterations and TDP-43 pathology in the degenerative process of the motor neurons of sALS patients.

Keywords: 43-kDa TAR DNA-binding protein; nuclear pore complex; nucleoporin; sporadic amyotrophic lateral sclerosis.

Fig. 1. TDP-43 and NUP62 immunoreactivity in control and sALS spinal motor neurons. TDP-43 was localized to the nucleus (arrowheads) and NUP62 immunoreactivity was present along the nuclear membrane in control spinal motor neurons (A–H). Motor neurons with decreased nuclear TDP-43 (arrowhead) exhibited fine TDP-43-positive dots (arrow) within the cytoplasm in sALS (I–L). NUP62 was present in the disrupted nuclear membrane of sALS spinal motor neurons with apparent TDP-43 mislocalization (arrows, arrowhead) (M–P). NUP62 immunoreactivity was absent (arrowheads) in some of the atrophic sALS spinal motor neurons with apparent TDP-43 mislocalization (arrows) (Q–T). Scale bars indicate 30 m. ^*Lipofuscin. DAPI: 4′,6-diamidino-2-phenylindole, NUP62: nucleoporin p62, sALS: sporadic amyotrophic lateral sclerosis, TDP-43: 43-kDa TAR DNA-binding protein.

Fig. 2. TDP-43 and KPNB1 localization in control and ALS spinal motor neurons. TDP-43 was localized in the nucleus (arrowhead), and KPNB1 labeling was present along the nuclear membrane and appeared as dots in the nucleus of control spinal motor neurons (A–D). KPNB1 labeling was present in the disrupted or deformed nuclear membrane of sALS spinal motor neurons with TDP-43 mislocalization (arrows, arrowheads) (E–L). Scale bars indicate 30 m. DAPI: 4′,6-diamidino-2-phenylindole, KPNB1: karyopherin beta 1, sALS: sporadic amyotrophic lateral sclerosis, TDP-43: 43-kDa TAR DNA-binding protein.

Fig. 3. Quantification of spinal motor neurons in lumbar spinal-cord tissue from control and sALS cases using double immunofluorescence labeling of NUP62 antibodies and TDP-43 antibodies. There were fewer AHC in sALS patients (52.2±36.4) than in control subjects (104.6±15.8) (p=0.015). Spinal motor neurons in control spinal anterior horns exhibited nuclear membranes that were labeled with anti-NUP62 antibodies and did not show TDP-43 mislocalization. In sALS spinal anterior horns, approximately two-thirds of the motor neurons exhibited normal round membranes without TDP-43-positive cytoplasmic inclusions. The remaining 34.8% of neurons in sALS samples exhibited TDP-43 mislocalization: 3.8% exhibited normal nuclear NUP62 immunoreactivity, 29.3% demonstrated disrupted NUP62 nuclear staining, and 2.7% were negative for NUP62 immunoreactivity. AHC: anterior horn cells, NUP62: nucleoporin p62, sALS: sporadic amyotrophic lateral sclerosis, TDP-43: 43-kDa TAR DNA-binding protein.