Regulation of nonphosphorylated and phosphorylated forms of neurofilament proteins in the prefrontal cortex of human opioid addicts

The neurofilament (NF) proteins (NF-H, NF-M, and NF-L for high, medium, and low molecular weights) play a crucial role in the organization of neuronal shape and function. In a preliminary study, the abundance of total NF-L wa s shown to be decreased in brains of opioid addicts. Because of the potenti al relevance of NF abnormalities in opioid addiction, we quantitated nonpho sphorylated and phosphorylated NF in postmortem brains from 12 well-defined opioid abusers who had died of an opiate overdose (heroin or methadone). L evels of NF were assessed by immunoblotting techniques using phospho-indepe ndent and phospho-dependent antibodies, and the relative (% changes in immu noreactivity) and absolute (changes in ng NF/mu g total protein) amounts of NF were calculated. Decreased levels of nonphosphorylated NF-H (42-32%), N F-M (14-9%) and NF-L (30-29%) were found in the prefrontal cortex of opioid addicts compared with sex, age, and postmortem delay-matched controls. In contrast, increased levels of phosphorylated NF-H (58-41%) and NF-M (56-28% ) were found in the same brains of opioid addicts. The ratio of phosphoryla ted to nonphosphorylated NF-H in opioid addicts (3.4) was greater than that in control subjects (1.6). In the same brains of opioid addicts, the level s of protein phosphatase of the type 2A were found unchanged, which indicat ed that the hyperphosphorylation of NF-H is not the result of a reduced dep hosphorylation process. The immunodensities of GFAP (the specific glial cyt oskeletol protein), alpha-internexin (a neuronal filament related to NF-L) and synaptophysin (a synapse-specific protein) were found unchanged, sugges ting a lack of gross changes in glial reaction, other intermediate filament s of the neuronal cytoskeletol, and synaptic density in the prefrontal cort ex of opioid addicts. These marked reductions in total NF proteins and the aberrant hyperphosphorylation of NF-H in brains of opioid addicts may play a significant role in the cellular mechanisms of opioid addiction. (C) 2000 Wiley-Liss, Inc.