RESUMO
The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.
RESUMO
Objective To investigate the relationship between protein phosphatase inhibition, tau hyperphosphorylation and neuronal death seen in Alzheimer disease (AD). Methods Co culture of protein phosphatase inhibitor okadaic acid (OA) and neuroblastoma cells (SH SY5Y), by agarose gel electrophoresis to detect DNA fragmentation, and in situ hybridization by TdT mediated biotin labeled dNTP nick end labeling (TUNEL) to further detect the cell apoptosis. Results Incubation of SY5Y cells with 10 nmol/L OA for 24 or 48 hours led to the appearance of DNA fragmentation and a remarkable increase of positive cells from 2 16%?0 94% to 18 05%?3 57% ( P
