The broad, long-term objective of this project is to elucidate the molecular mechanisms of action of environmental and occupational neurotoxins which induce axonal degeneration in experimental animals and man. This proposal examines an important subclass of this group, the neurofilament (NF) neurotoxins, which includes n-hexane, 2,5-hexanedione (2,5-HD), carbon disulfide (CS2), beta, beta-iminodipropionitrile (IDPN), an acrylamide. These agents produce cytoskeletal disorganization and NF accumulation within susceptible nerve fibers. The NF neurotoxins represent a significant public health hazard, and in view of their great chemical and structural diversity, it is important to identify common steps in their respective mechanisms. Such information would be of great value in predicting similar effects from untested or newly synthesized chemicals.
Specific aims i nclude: i) Characterization and quantitation of the binding of 2,5-HD, CS2, IDPN (or its metabolites), and acrylamide to axonal cytoskeletal proteins; ii) Determination of whether such derivatization is limited to specific sites within the NF proteins or is randomly distributed; iii) Elucidation of the effects of in vitro and in vivo neurotoxin exposure on NF-NF and NF-microtubule (MT) interactions and on axonal cytoskeletal structures; and iv) Determination of the differential susceptibility of the distal vs. proximal axon to uptake of and covalent derivatization by NF neurotoxins. In vitro axonal cytoskeletal and whole animal model systems will be employed. Binding studies will utilize high specific activity [14C]-labelled NF neurotoxins. Analytical techniques will include gel electrophoresis of cytoskeletal proteins, fluorography, and scintillation counting of gel slices. Sites of protein binding will be assessed by chemical and enzymatic cleavage followed by HPLC peptide mapping and automated peptide sequencing. Protein adducts will be characterized by mass spectrometry. NF-NF interactions will be assessed by measuring the kinetics of NF protein reassembly and NF network formation using native NFs from treated rats. NF-MT affinity will be studied with blotting assays utilizing isolated NFs and [32P]-labelled MTs. Cytoskeletal reorganization will be examined in organotypic nerve cultures using video-enhanced and differential interference contrast light microscopy, immunofluorescence microscopy, and high voltage electron microscopy. Regional uptake and covalent binding of radiolabelled NF neurotoxins will be assessed along the optic nerve of treated rats and in organotypic nerve cultures. Findings will provide substantial progress towards defining mechanisms of action for this important class of neurotoxins.
| LoPachin, Richard M; DeCaprio, Anthony P (2004) gamma-Diketone neuropathy: axon atrophy and the role of cytoskeletal protein adduction. Toxicol Appl Pharmacol 199:20-34 |
