Phosphorylation of neurofilament H subunit as related to arrangement of neurofilaments

Abstract

To find out what causes differences in phosphorylation states in neurofilaments (NF), we selected two types of dendrite, one provided with very few NFs (Purkinje cell) and the other with relatively many (anterior horn cell). We examined these with four monoclonal antibodies selected by the Western blot analysis, two (NE14 and SMI31) recongnizing only phosphorylated, SMI32 recognizing only nonphosphorylated, and N52 recognizing phosphorylation‐independent epitopes of NF‐H. The immunoperoxidase labeling of dendrites, and also of perikarya, in both neurons was detectable with all four antibodies. After the tissue was treated with Triton X‐100, the labeling was still detectable with SMI32 or N52, but undetectable with NE14 and SMI31. The brain homogenate Triton‐extracted supernatant after centrifugation at 100,000g for 1 hr showed the staining of NE14, SMI31, and N52 but not that of SMI32. In Purkinje cell dendrite and perikaryon, NFs always appeared singly. In the immunogold labeling, they were labeled only with SMI32 or N52. Labeling by NE14 or SMI31 was distributed throughout the cytoplasm and hardly associated with NFs. In the anterior horn cell dendrite and perikaryon, NFs appeared both single and in bundles. They were predominantly labeled with SMI31 or N52 when they were single, and with NE14, SMI31, or N52 when they were bundled. Even in one NF, portions that appeared single were labeled mostly with SMI32 or N52, while the remainder, to which other NFs approached closely, were labeled mostly with NE14, SMI31, or N52. Thus, when NFs appear singly, NF‐H in their projections or cross‐bridges with other organelles is not phosphorylated, while when NFs are bundled, NF‐H is phosphorylated in crossbridges between NF core filaments. These data may explain why the NF‐H is heavily phosphorylated in axons, where NFs are abundant, and not in dendrites and perikarya, where NFs are sparse. Wiley‐Liss, Inc.