One of the prevalent pathologies in Alzheimer's Disease is the accumulation of ubiquitin-immunoreactive material in cell bodies, dendrites, and neurites. The neuritic pathology is recapitulated in transgenic mouse models of beta-amyloid deposition. The prevalence of this pathology in AD, as well as other disorders of the CNS, has led to the hypothesis that a dysfunction of the ubiquitin/proteasome system may play a role in the pathogenesis of disease. However, probing the function of this system in vivo is problematic and it has been difficult to know whether dysfunction of this system is an early or late event. We propose to produce transgenic mice that express proteasome sensors. We will construct two sensors by fusing the coding sequence of ubiquitin (encoding mutations that preclude endoproteolytic processing) to green fluorescent protein and beta-galactosidase. These proteins will be short- lied in normal settings. We will insert these cDNAs into the mouse PrP vector and generate transgenic mice. We will then screen for lines of mice in which expression of mRNA for the sensor is high, while the accumulated levels of protein are negligible. We will then generate primary neuronal cultures and use chemical inhibitors of the proteasome to screen for mice in which inhibition of the proteasome leads to increased accumulation of our sensor proteins. Ultimately, if successful, these animals would provide function in normal aging, and in our transgenic mouse models of Alzheimer's Disease and other neurodegenerative diseases.
