Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis.
OBJECTIVE
TAR DNA-binding protein of 43kDa (TDP-43) is deposited as cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43.


METHODS
We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy, and immunoblots. In addition, we performed investigations aimed at identifying the responsible kinases, and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization.


RESULTS
We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially available anti-TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15nm diameter and on immunoblots recognized hyperphosphorylated TDP-43 at 45kDa, with additional 18 to 26kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase-1 and -2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43.


CONCLUSIONS
These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders.
Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo, Japan. masato@prit.go.jp
Masato
M
Hasegawa
Tetsuaki
T
Arai
Takashi
T
Nonaka
Fuyuki
F
Kametani
Mari
M
Yoshida
Yoshio
Y
Hashizume
Thomas G
TG
Beach
Emanuele
E
Buratti
Francisco
F
Baralle
Mitsuya
M
Morita
Imaharu
I
Nakano
Tatsuro
T
Oda
Kuniaki
K
Tsuchiya
Haruhiko
H
Akiyama
D000368:Aged; D000369:Aged, 80 and over; D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism* / Q000473:pathology*; D000906:Antibodies; D000918:Antibody Specificity; D001921:Brain / Q000378:metabolism / Q000473:pathology*; D047389:Casein Kinase I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D003704:Dementia / Q000378:metabolism* / Q000473:pathology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000276:immunology / Q000473:pathology; D008297:Male; D008875:Middle Aged; D016874:Neurofibrillary Tangles / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary
D000906:Antibodies; D004268:DNA-Binding Proteins; D000939:Epitopes; D010446:Peptide Fragments; D047389:Casein Kinase I