Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster.

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA with a two-base deletion. If the deletion occurs in a coding region, translation will result in production of misframed proteins. During mammalian aging, misframed versions of human amyloid precursor protein (hApp) and ubiquitin (hUbb) accumulate in the aggregates characteristic of neurodegenerative diseases, suggesting dysfunctional degradation or clearance. Here cDNA clones encoding wild-type hUbb and the frame-shifted version hUbb(+1) were expressed in transgenic Drosophila using the doxycycline-regulated system. Misframed proteins were abundantly produced, both from the transgenes and from endogenous Drosophila ubiquitin-encoding genes, and their abundance increased during aging in whole-fly extracts. Over-expression of wild-type hUbb, but not hUbb(+1), was toxic during fly development. In contrast, when over-expressed specifically in adult flies, hUbb(+1) caused small decreases in life span, whereas hUbb was associated with small increases, preferentially in males. The data suggest that MM occurs in Drosophila and that the resultant misframed proteins accumulate with age. MM of the ubiquitin gene can produce alternative ubiquitin gene products with different and sometimes opposing phenotypic effects.

Alzheimer-associated mutant ubiquitin impairs spatial reference memory.

UBB(+1) is a mutant ubiquitin which accumulates in the hallmarks of tauopathies, including Alzheimer's disease. Transgenic mice expressing high levels of neuronal UBB(+1) exhibit moderately decreased proteasome activity and spatial reference memory deficits at 9months of age. In the present study, we characterized the behavioral phenotype of male UBB(+1) transgenic mice at different ages. We show that UBB(+1) transgenic mice displayed an age-related functional decline similar to wild-type littermates, without gross neurological abnormalities or alterations in procedural motor-learning and motor coordination. At 15months of age, a transgene-specific spatial learning deficit was dependent on the period of training in the Morris watermaze. This deficit could be eliminated after additional training. We conclude that the previously reported spatial reference memory deficits of UBB(+1) transgenic mice persist during aging. In addition, our results demonstrate that the subtle defect in spatial reference memory formation, caused by a decrease in forebrain proteasome activity, is a persistent defect and not a structural defect.

Low levels of mutant ubiquitin are degraded by the proteasome in vivo.

The ubiquitin-proteasome system fulfills a pivotal role in regulating intracellular protein turnover. Impairment of this system is implicated in the pathogenesis of neurodegenerative diseases characterized by ubiquitin- containing proteinaceous deposits. UBB(+1), a mutant ubiquitin, is one of the proteins accumulating in the neuropathological hallmarks of tauopathies, including Alzheimer's disease, and polyglutamine diseases. In vitro, UBB(+1) properties shift from a proteasomal ubiquitin-fusion degradation substrate at low expression levels to a proteasome inhibitor at high expression levels. Here we report on a novel transgenic mouse line (line 6663) expressing low levels of neuronal UBB(+1). In these mice, UBB(+1) protein is scarcely detectable in the neuronal cell population. Accumulation of UBB(+1) commences only after intracranial infusion of the proteasome inhibitors lactacystin or MG262, showing that, at these low expression levels, the UBB(+1) protein is a substrate for proteasomal degradation in vivo. In addition, accumulation of the protein serves as a reporter for proteasome inhibition. These findings strengthen our proposition that, in healthy brain, UBB(+1) is continuously degraded and disease-related UBB(+1) accumulation serves as an endogenous marker for proteasomal dysfunction. This novel transgenic line can give more insight into the intrinsic properties of UBB(+1) and its role in neurodegenerative disease.

Long-term proteasome dysfunction in the mouse brain by expression of aberrant ubiquitin.

Many neurodegenerative diseases are characterized by deposits of ubiquitinated and aberrant proteins, suggesting a failure of the ubiquitin-proteasome system (UPS). The aberrant ubiquitin UBB(+1) is one of the ubiquitinated proteins accumulating in tauopathies such as Alzheimer's disease (AD) and polyglutamine diseases such as Huntington's disease. We have generated UBB(+1) transgenic mouse lines with post-natal neuronal expression of UBB(+1), resulting in increased levels of ubiquitinated proteins in the cortex. Moreover, by proteomic analysis, we identified expression changes in proteins involved in energy metabolism or organization of the cytoskeleton. These changes show a striking resemblance to the proteomic profiles of both AD brain and several AD mouse models. Moreover, UBB(+1) transgenic mice show a deficit in contextual memory in both water maze and fear conditioning paradigms. Although UBB(+1) partially inhibits the UPS in the cortex, these mice do not have an overt neurological phenotype. These mouse models do not replicate the full spectrum of AD-related changes, yet provide a tool to understand how the UPS is involved in AD pathological changes and in memory formation.

Disease-specific accumulation of mutant ubiquitin as a marker for proteasomal dysfunction in the brain.

Molecular misreading of the ubiquitin-B (UBB) gene results in a dinucleotide deletion in UBB mRNA. The resulting mutant protein, UBB+1, accumulates in the neuropathological hallmarks of Alzheimer disease. In vitro, UBB+1 inhibits proteasomal proteolysis, although it is also an ubiquitin fusion degradation substrate for the proteasome. Using the ligase chain reaction to detect dinucleotide deletions, we report here that UBB+1 transcripts are present in each neurodegenerative disease studied (tauo- and synucleinopathies) and even in control brain samples. In contrast to UBB+1 transcripts, UBB+1 protein accumulation in the ubiquitin-containing neuropathological hallmarks is restricted to the tauopathies such as Pick disease, frontotemporal dementia, progressive supranuclear palsy, and argyrophilic grain disease. Remarkably, UBB+1 protein is not detected in the major forms of synucleinopathies (Lewy body disease and multiple system atrophy). The neurologically intact brain can cope with UBB+1 as lentivirally delivered UBB+1 protein is rapidly degraded in rat hippocampus, whereas the K29,48R mutant of UBB+1, which is not ubiquitinated, is abundantly expressed. The finding that UBB+1 protein only accumulates in tauopathies thus implies that the ubiquitin-proteasome system is impaired specifically in this group of neurodegenerative diseases and not in synucleinopathies and that the presence of UBB+1 protein reports proteasomal dysfunction in the brain.