Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis

Ann Neurol. 2008 Jul;64(1):60-70. doi: 10.1002/ana.21425.

Abstract

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.

Interpretation: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Amyotrophic Lateral Sclerosis / metabolism*
  • Amyotrophic Lateral Sclerosis / pathology*
  • Antibodies
  • Antibody Specificity
  • Brain / metabolism
  • Brain / pathology*
  • Casein Kinase I / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / immunology
  • DNA-Binding Proteins / metabolism*
  • Dementia / metabolism*
  • Dementia / pathology*
  • Epitopes / chemistry
  • Epitopes / immunology
  • Female
  • Humans
  • Inclusion Bodies / chemistry
  • Inclusion Bodies / immunology
  • Inclusion Bodies / pathology
  • Male
  • Middle Aged
  • Neurofibrillary Tangles / immunology
  • Neurofibrillary Tangles / metabolism
  • Neurofibrillary Tangles / pathology
  • Peptide Fragments / chemistry
  • Peptide Fragments / immunology
  • Phosphorylation
  • Protein Structure, Tertiary

Substances

  • Antibodies
  • DNA-Binding Proteins
  • Epitopes
  • Peptide Fragments
  • Casein Kinase I