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.

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.

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.

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