TDP-43 redistribution is an early event in sporadic amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder consisting of progressive loss of motor neurons. TDP-43 has been identified as a component of ubiquitin-immunoreactive inclusions of motor neurons in ALS. We focused on the diffuse cytoplasmic TDP-43 immunoreactivity in ALS neurons, and quantitatively assessed it in comparison with skein/round TDP-43 and ubiquitin immunostaining in motor neurons of 30 sporadic ALS cases. The percentage of spinal motor neurons with cytoplasmic TDP-43 immunoreactivity was higher than that of ubiquitin-immunoreactive ones. The percentage of TDP-43-positive motor neurons was independent of neuron counts in anterior horns, while the percentage of ubiquitinated neurons was inversely correlated. Aiming to define the cytosolic localization of TDP-43, the immunoblot analysis of spinal cord and frontal cortex showed that full-length TDP-43, the 45 kDa form and ubiquitinated TDP-43 are found in the soluble inclusion-free fraction. The present data suggest that delocalization, accumulation and ubiquitination of TDP-43 in the cytoplasm of motor neurons are early dysfunctions in the cascade of the events leading to motor neuron degeneration in ALS, preceding the formation of insoluble inclusion bodies. Being cytoplasmic accumulation an ongoing event during the course of the illness, a therapeutic approach to this incurable disease can be envisaged.

Figure 1

Characterization of pellets A and B, and cytosolic fraction from samples of amyotrophic lateral sclerosis spinal cord (ALS) and controls (ctrl) by using the nuclear‐specific markers lamin B1 and histone H4. Pellets A and B are positive to the nuclear markers; the cytosolic fraction [cytosolic fraction free from insoluble material (CS‐F)] is negative.

Figure 2

TDP‐43 and ubiquitin immunohistochemistry in sporadic ALS. (A) TDP‐43‐ and (B) ubiquitin‐positive skein‐like inclusion in spinal motor neurons; the nuclei of neurons are TDP‐43‐negative, DAB; (C), (D), (E), (G), (H), (I) Diffuse cytoplasmic TDP‐43 immunoreactivity in spinal motor neurons; (F) no ubiquitin staining in the TDP‐43‐positive neuron shown in (E), DAB; (J), (K) TDP‐43‐positive cytoplasm of neurons in the frontal cortex, DAB. Scale bars = 20 µm.

Figure 3

Double‐label immunofluorescence of cytoplasmic inclusions in an anterior horn motor neuron of a sporadic amyotrophic lateral sclerosis patient: TDP‐43 (A) and ubiquitin (B) do not completely overlap (C). A. Ubiquitin: TRIC, red. B. TDP‐43: FITC, green. C. Merged. ×1000.

Figure 4

Percentage of TDP‐43‐ and ubiquitin‐positive spinal motor neurons by number of residual motor neurons in anterior horns of 30 sporadic amyotrophic lateral sclerosis patients.

Figure 5

Percentage of TDP‐43‐ and ubiquitin‐ positive dentate neurons of hippocampus in the six immunopositive amyotrophic lateral sclerosis cases.

Figure 7

Immunoblotting of the cytosolic fractions (CS‐F) from samples of spinal cord (A,B) and cerebral cortex (C,D) of amyotrophic lateral sclerosis (ALS) and control patients (ctrl) with antibodies to TDP‐43 and ubiquitin. Arrowheads: 43 kDa, normal TDP‐43; 45 kDa: pathological form of TDP‐43. GAPDH immunoreactivity was used to confirm equal protein loading. The images are representative of three independent experiments performed on three patients and three controls.

Figure 6

Immunoblotting of the sarkosyl‐insoluble fraction (SarkI‐P) from spinal cord specimens of amyotrophic lateral sclerosis patients (ALS) and control (ctrl) with antibodies to TDP‐43 (A) and ubiquitin (B). The 43 kDa band corresponds to full‐length TDP‐43; the 45 and 28 kDa bands are pathological TDP‐43 forms; the TDP‐43‐immunostained protein bands below 43 kDa are presumably cleavage products of TDP‐43 (29). (*) High‐molecular mass smear.