DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of beta-tubulin subtypes in the CNS of hens contributes to OPIDN.
Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130+/-5%) and 8 h (133+/-6 %), while cerebrum nuclear fraction showed decrease (77+/-5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133+/-3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150+/-3%) and 5 days (173+/-7%) of treatment, in contrast to the decreased levels p-CREB (72+/-4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did not show any changes in pCREB at time points studied. Similarly another set of animals were treated with DFP and perfused using standard protocols and immunohistochemistry for p-CREB in the brain and spinal cord confirmed the overall protein expression pattern identified by western analysis. Expression of beta-tubulin subtypes (1, 2, 3, and 4), studied by Northern blotting showed complex and differential pattern, while immunohistochemistry of the anti-beta-tubulin for the entire period of OPIDN developmental stages showed early induction and persistence even in the disintegrating axonal and non-neuronal structures of the CNS. These data thus strongly suggest that early cytoskeletal damage at molecular level mediated by PKA/p-CREB pathways leads to the culmination of gross (microscopically observable) level cytoskeletal changes in various components of central nervous system (CNS), consistent with our earlier findings. Thus, the differential protein expression of PKA, CREB, p-CREB and beta-tubulin subtypes appear to contribute to the initiation, progression and development of OPIDN, probably by recruiting other molecular pathways specific to various components of nervous system.
Damodaran
Tirupapuliyur V
TV
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Gupta
Ram P
RP
Attia
Moustafa K
MK
Abou-Donia
Mohamed B
MB
D000818:Animals; D000931:Antidotes / Q000494:pharmacology; D001285:Atropine / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D002490:Central Nervous System / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D054022:Cerebrum / Q000187:drug effects / Q000201:enzymology; D002645:Chickens; D002800:Cholinesterase Inhibitors / Q000633:toxicity*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018450:Disease Progression; D005260:Female; D007150:Immunohistochemistry; D007531:Isoflurophate / Q000633:toxicity*; D019636:Neurodegenerative Diseases / Q000139:chemically induced* / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control; D020258:Neurotoxicity Syndromes / Q000201:enzymology / Q000209:etiology* / Q000473:pathology / Q000517:prevention & control; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013116:Spinal Cord / Q000187:drug effects / Q000201:enzymology; D013997:Time Factors; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D000931:Antidotes; D002800:Cholinesterase Inhibitors; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D014404:Tubulin; D007531:Isoflurophate; D001285:Atropine; D017868:Cyclic AMP-Dependent Protein Kinases