title: Effect of Alzheimer's brain extracts on dynein immunoreactivity in PC12 cells.
abstract: The neurodegenerative process in Alzheimer's disease (AD) has been suggested to occur as a consequence of microtubule disruption and subsequent loss of intracellular transport. Structural microtubule-associated proteins (MAPs) have been investigated for their role in the etiology of AD, but dynein, a force-producing MAP which mediates intracellular transport, has not been examined. In this report, dynein (MAP1C) immunoreactivity in AD brain tissue homogenates was observed increased 3.7-fold compared with control brain homogenate preparations. Similarly, NGF-differentiated PC12 cells cultured in the presence of soluble extracts prepared from AD brain tissue homogenates, exhibited an approximate 15-fold increase in dynein immunoreactivity compared to that of control brain tissue extracts. In contrast, AD clarified extracts had little effect upon "kinesin-like" protein immunoreactivity increased (approximately 2-fold); whereas, tau immunoreactivity was observed to be moderately increased (5-fold) over that of control brain extract treated PC12 cells. Chemical dephosphorylation and alkaline phosphatase treatment of AD extract-treated PC12 cell lysate prior to Western blotting resulted in complete loss of immunoreactivity, suggesting the dynein being monitored is a phosphorylated isoform. Furthermore, treatment of clarified brain tissue extracts with trypsin and (NH4)2SO4 suggests the endogenous elements giving rise to increased PC12 cell dynein intermediate chain immunoreactivity to be proteinaceous in nature. The observed increase in dynein intermediate-chain dynein immunoreactivity following exposure of neuronal cells to endogenous elements of AD brain may be reflective of dynein-microtubular array differences. Such an approach may be useful in assessing the effect of endogenous biomolecules on retrograde axonal transport in neuronal culture models.
authors.affiliation: Division of Life Sciences, University of Texas at San Antonio, 78249, USA.
authors.forename: K
authors.initials: K
authors.lastname: Kopec
authors.forename: J P
authors.initials: JP
authors.lastname: Chambers
mesh_terms: D000544:Alzheimer Disease / Q000276:immunology / Q000378:metabolism*; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002452:Cell Count; D002458:Cell Fractionation; D004398:Dyneins / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016547:Kinesins / Q000737:chemistry / Q000276:immunology; D008928:Mitochondria / Q000201:enzymology; D010088:Oxidoreductases / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D014020:Tissue Extracts / Q000302:isolation & purification / Q000502:physiology; D014357:Trypsin / Q000494:pharmacology; D016875:tau Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D014020:Tissue Extracts; D016875:tau Proteins; D010088:Oxidoreductases; D014357:Trypsin; D004398:Dyneins; D016547:Kinesins; D000645:Ammonium Sulfate