“The effect of mutant ubiquitin on proteasome function in relation to neurodegenerative disease”

Paula van Tijn SUMMARY

The ubiquitin-proteasome system (UPS) is the main intracellular regulated pathway for degradation of substrate proteins and, being one of the main cellular protein quality control systems, is essential for cell viability and maintaining proteostasis. Malfunction of this system is implicated to play a role in a broad array of diseases pathologically characterized by ubiquitin-positive deposits. The aberrant ubiquitin UBB +1 is present in the neuropathological hallmarks of a subset of these diseases, including Alzheimer's disease and Huntington's disease, providing compelling evidence that UPS malfunction can contribute to the pathological cascade leading to (neuro-) pathology. in vitroin vivo, employing novel UBB +1 transgenic mouse models. Chapter 1 gives a comprehensive overview of the UPS and its role in neurodegenerative disease. In addition, the currently available murine models with altered components of the UPS leading to neurological deficits are reviewed.

The aim of the research described in Chapter 2 was to further characterize the UPS-related properties of UBB +1 . UBB +1 has seemingly opposing properties in vitro; UBB +1 is a substrate for proteasomal degradation as well as an inhibitor of the UPS. In this study, we showed that UBB +1 properties shift from proteasome substrate to inhibitor in a dose-dependent manner in cell culture using an inducible UBB +1 expression system. This finding was confirmed in mouse organotypic cortex slice cultures. Based on this study combined with previous findings, we hypothesize that in the human brain, once the level of UBB +1 protein has surpassed a threshold level of expression, UBB +1 induced UPS inhibition can contribute to disease progression. Using the dual UPS proteasome substrate/inhibitory properties, UBB +1 can serve as research tool to study the ubiquitin-proteasome system and to further elucidate the role of aberrations of this pathway in disease.

In Chapter 3, the effects of high levels of UBB +1 expression are studied in vivo. To this end, two transgenic mouse lines were generated, which postnatally express high levels of UBB +1 under the neuronal CaMKinaseIIa (line 3413) or the Thy1.2 (line 8630) promoter. In both lines, UBB +1 protein expression was present mainly in the cortex and hippocampus. Increased levels of ubiquitinated proteins were detected in the cortex, suggesting inhibition of the UPS. Indeed, chymotryptic proteasome activity was decreased in the cortex of line 3413 mice. Despite this low-level chronic UPS inhibition, these mice did not show an overt neurological phenotype. However, deficits in contextual memory in both Morris watermaze and fear conditioning paradigms were present at the age of 9 months. Furthermore, proteomic analysis of these mice showed a remarkable overlap with changes in the brain proteome reported in Alzheimer's patients and in Alzheimer mouse models. These UBB +1 transgenic mouse models provide new tools to understand how the UPS is involved in neurodegenerative pathology and memory formation. In addition, these UBB +1 transgenic lines serve as model for life-long neuronal modest UPS inhibition.

Chapter 4 describes the generation and characterization of a transgenic mouse model (line 6663) neuronally expressing low levels of UBB +1 . In this mouse line, UBB +1 protein was detected at very low levels. Via intracranial infusion of different classes of proteasome inhibitors into the hippocampus, we showed that UBB +1 protein accumulated only when proteasome inhibitor was administrated. These in vivo results confirm our previous in vitrodata, as presented in Chapter 2, showing that UBB +1 is a substrate for proteasomal degradation at low expression levels and only accumulates after inhibition of the UPS. This transgenic mouse model can serve as a model system to further elucidate the properties of UBB +1 and to study its role in neurodegenerative disease. Furthermore, this mouse model can serve as a reporter line for UPS inhibition associated with disease, employing a natural substrate rather than the widely used artificial fluorescent reporters currently used by the research community.

Chapter 5 describes the effect of low-level UPS inhibition of cognitive function. The UPS plays an important role in synaptic plasticity and learning and memory formation in the adult nervous system. We hypothesized that proteasome inhibition induced by UBB +1 expression would thus lead to a decreased cognitive function. Indeed, we showed that UBB +1 transgenic mice showed a defect in spatial reference memory in the Morris watermaze at 15 months of age. The UBB +1 transgenic mice did not display further gross neurological abnormalities or alterations in procedural (motor-) learning and motor coordination up to 24 months of age. From these results, we conclude that the spatial reference memory deficits detected in UBB +1 transgenic mice at 9 months, as described in Chapter 3, persist, but are not aggravated during aging. In addition, these results demonstrate that intact forebrain proteasome function is essential for maintenance of spatial reference memory formation.

Chapter 6 reports on the effects of UBB +1 induced proteasome inhibition on Alzheimer related neuropathology, i.e. Aß deposition, as well as the effects of Aß deposition on UBB +1 accumulation in vivo. In a novel triple transgenic mouse model, expressing UBB +1 and familial Alzheimer's disease related mutant APP and PS1, we show that modest neuronal UPS inhibition induced by UBB +1 expression reduced the amyloid plaque burden at the age of 6 months, without alterations in UBB +1 protein levels or in the age of onset of pathology. It is conceivable that APP processing, leading to Aß formation, is affected by proteasome inhibition, resulting in a decreased plaque burden.

Finally, in Chapter 7, the results obtained in the studies described in this thesis are critically discussed and suggestions for future research are provided. Additional preliminary results further elucidate the role of mutant ubiquitin and concomitant proteasome dysfunction in neurodegenerative disease.

Based on the findings as presented in this thesis, we conclude that accumulation of aberrant ubiquitin and subsequent malfunction of the UPS affects protein degradation in vitro and in vivo, can lead to spatial memory deficits and interferes with Alzheimer's disease-associated amyloid pathology. The novel transgenic mouse models expressing mutant ubiquitin, as presented in Chapter 3 and Chapter 4, can contribute to dissecting the role of the UPS in a broad range of neurological diseases in future studies.