Effect of Alzheimer's Brain Extracts on Dynein Immunoreactivity in PC12 Cells

Abstract

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, × 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.