| Abstract No.: | A-C1105 |
| Country: | Canada |
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| Title: | 4-HYDROXY-2-NONENAL IMPAIRS AXON OUTGROWTH, CAUSES ACCUMULATION OF MITOCHONDRIA IN ABERRANT AXONAL STRUCTURES AND MIMICS THE EFFECT OF DIABETES IN CULTURED ADULT SENSORY NEURONS |
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| Authors/Affiliations: | 2 Eli Akude*; 1 Elena Zherebitskaya; 2 Paul Fernyhough;
1 Div. of Neurodegenerative Disorders, St. Boniface Hospital Research Centre; 2 Div. of Neurodegenerative Disorders, St. Boniface Hospital Research Centre and Dept. of Pharmacology & Therapeutics, Winnipeg, MB, Canada
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| Content: | Objectives. Protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) have been associated with disease states involving oxidative stress. Modification of proteins by 4-HNE in peripheral nerve tissue occurs in diabetic sensory neuropathy, however, the target proteins and impact on nerve function are unknown. We, therefore, tested the ability of 4-HNE to induce amino acid adduct formation on axonal cytoskeletal proteins and determined if such modifications were associated with aberrant axon morphology, mitochondrial accumulation, and suboptimal axon outgrowth in cultured adult sensory neurons. The impact of type 1 diabetes on these processes was also investigated.
Methods. Adult rat dorsal root ganglion (DRG) sensory neurons were cultured in defined F12-media supplemented with neurotrophic factors (NTFs;1nM insulin, 1ng/ml NGF, 10ng/ml GDNF and 10ng/ml NT-3) and treated with 4-HNE concentrations ranging from 1.0µM to 10 µM. Cell survival, axonal morphology and level of axonal regeneration were assessed at 24 hours in culture. Western blot and immunofluorescent staining were utilized to detect protein adduct formation by 4-HNE (anti-4-HNE, Alexis Biochemicals) and phosphorylation levels of neurofilament H protein (antibody SMI31, Covance). Also, a mitochondrial specific dye (1.0 µM Mitotracker red CMXRos; Invitrogen) was utilized to detect the presence of mitochondrial accumulation in the aberrant structures along the axons.
Results. 4-HNE induced formation of amino acid adducts on neurofilament H protein and impaired axon regeneration by approximately 50% (ED50 3M) whilst having no effect on neuronal survival. 4-HNE initiated formation of aberrant axonal structures and caused the accumulation of mitochondria in these structures, which mimicked those seen in axons of neurons under diabetic conditions (in animal models and humans). The formation of protein adducts also led to diminished levels of phosphorylation of neurofilament H protein. Sensory neurons from 3 month streptozotocin-diabetic rats showed abnormal axonal swellings which were filled with 4-HNE protein adducts and impaired levels of axon outgrowth; control neurons exhibited negative staining for 4-HNE in axons.
Conclusions. This study demonstrates that 4-HNE induces amino acid adduct formation on neurofilament H protein, causes mitochondria accumulation, and this modification is associated with impairment of axonal regeneration. The results show that 4-HNE might be an important link between oxidative stress triggered lipid peroxidation and subsequent modification of key neuronal cytoskeletal and mitochondrial proteins in diabetic sensory neuropathy. It is proposed that 4-HNE mediated abnormalities in neurofilament and mitochondrial function, and possibly other cytoskeletal proteins, may cause distal axon degeneration through suboptimal mitochondrial motility and localization.
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