Synergistic effect between proteasome and autophagosome in the clearance of polyubiquitinated TDP-43.
Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS.
Unit for Neurobiology and Therapeutics, Molecular Neuroscience Research Center, Shiga University of Medical Science, Shiga, Japan. uru@belle.shiga-med.ac.jp
Makoto
M
Urushitani
Takashi
T
Sato
Hitoshi
H
Bamba
Yasuo
Y
Hisa
Ikuo
I
Tooyama
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000704:Analysis of Variance; D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection / Q000379:methods; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; C485184:enhanced green fluorescent protein; C067713:lactacystin; D049452:Green Fluorescent Proteins; C025946:3-methyladenine; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000225:Adenine; D000111:Acetylcysteine