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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. Ann Neurol 2008