Onset and Progression of Neurodegeneration in Mouse Models for Defective Endocytosis
by Christine Melina Rostosky
Date of Examination:2018-11-09
Date of issue:2019-10-25
Advisor:Dr. Ira Milosevic
Referee:Dr. Ira Milosevic
Referee:Prof. Dr. Thomas A. Bayer
Referee:Prof. Dr. Tiago Fleming Outeiro
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Abstract
English
Neurodegenerative diseases have become an increasing problem in our aging society. Many studies have addressed these complex diseases in the recent years. Scientists have learned a lot about specific proteins, their regulation and roles in diseases, yet there are still many unanswered questions in this area. As the knowledge increases, it has become apparent that these diseases affect more than one protein and that entire processes are dysregulated or dysfunctional, leading to similar but often non-identical symptoms in patients of different neurodegenerative diseases. Emerging evidence is accumulating that while the proteins involved in these diseases are diverse, many of them ultimately disrupt basic cellular processes in neurons. It is therefore not surprising that recent reports have shown that impairments of endocytosis or autophagy, two processes involved in maintaining proteostasis in synapses, can lead to neurodegeneration. This thesis was designed to shed light on questions of if and how the family of endophilin-As, key proteins in synaptic endocytosis, is involved in neurodegeneration. Taking advantage of the complex endophilin mouse model, this thesis shows that endophilin mutants have progressive motor impairments depending on the number of deleted endophilin alleles and the animal age. The motor deficits are prominently detected in the Rotarod test which requires motor coordination and in the overall running ability on a motorized treadmill and subtly found in gait analysis or general muscle strength. Furthermore, data presented here also reveal signs of neurodegeneration in mouse models for endophilin deficiency. The evidence points to a mild form of neurodegeneration that is found in the hippocampus, cortex and cerebellum. These findings include an increase in apoptotic and necrotic cells in brain sections from different endophilin mutants and a strong gliosis in endophilin 1,2-DKO mice. Unexpectedly, data reported in this thesis, complemented by additional data produced in the laboratory, indicate that endophilin has a previously unknown role in autophagy in addition to its well-described role in endocytosis. Most prominently, an E3 ubiquitin ligase, Fbxo32, is robustly up-regulated in various endophilin mutants. Fbxo32 has previously been suggested to act as a mediator between the ubiquitin-proteasome system and autophagy. Congruously, endophilin mutants are overloaded with poly-ubiquitinated proteins that are marked for degradation through the UPS and have decreased levels of Atg5, a protein involved in the formation of the autophagophore. However, because of this double role of endophilin, it is challenging to discriminate between the impact of endocytosis and autophagy impairments on the systemic level. In order to answer this question, two additional mouse models for defective endocytosis, synaptojanin-1 and dynamin 1,3 deficient mice, were also tested for motor impairments and the regulation of autophagy markers. In this study, no motor impairments were discovered in aged synaptojanin-1 deficient mice and only mild motor deficits in dynamin mutants. In addition, the amount of poly-ubiquitinated proteins and Atg5 were unchanged in synaptojanin and dynamin deficient mice. To elucidate how defective autophagy and endocytosis impact motor behavior in the endophilin mouse model, this thesis attempted to normalize altered proteostasis in the endophilin mutants by genetically reducing the levels of Fbxo32. In fact, motor deficits were ameliorated in endophilin mutants who are also heterozygous for Fbxo32. Taken together, this work sheds light on endophilin’s role in endocytosis and autophagy and the increasing importance of protein homeostasis on neuronal survival.
Keywords: Neurodegeneration; Endophilin; Endocytosis; Fbxo32; Motor Behavior; endophilin-A; E3 ubiquitin ligase; autophagy; synaptojanin; dynamin; mouse model; motor coordination; gait; rotarod; aging