Connecting protein aggregation and granulovacuolar degeneration bodies

The aggregation of specific proteins in the human brain is at the core of neurodegenerative disorders such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). Despite being the best correlate for neuronal death in AD patients, the effects of tau misfolding and aggregation in neurons are not completely understood. This thesis investigates changes in the neuronal lysosomal system caused by pathological protein assemblies with a focus on tau and granulovacuolar degeneration bodies (GVBs), a neuron-specific lysosomal structure associated with tau pathology. In Chapter 2, we studied the relationship between protein aggregation and GVB formation. Our data demonstrate that in the context of tau pathology, GVBs are inseparably associated with pathological tau accumulation at the cellular level. This was shown in human post mortem tissue and a newly developed primary neuron model for tau aggregation. The model was also used to study the sequence of events, demonstrating that pathological tau accumulation precedes GVB formation. Last, we tested whether α-syn aggregation can also induce GVB formation. The association between GVBs and pathological accumulation of α-syn was demonstrated both in the brain of PD patients and in an α-syn aggregation model in primary neurons in the absence of cytosolic accumulation of pathological tau. GVBs associated with tau and α-syn aggregates have a similar immunoreactivity signature, including phosphorylated tau epitopes. Our data indicate that GVBs occur as a more generalized response to the accumulation of pathological protein assemblies in neurons. The aim of Chapter 3 was to investigate factors in the aggregation of tau that could modulate GVB formation. To this end, the aggregates of untagged and GFP-tagged variants of the double mutant tau in our primary neuron model were characterized. We demonstrate that the resulting GFP-tagged pathological tau assemblies have a different morphology, ultrastructure and subcellular localization compared to untagged tau. Untagged tau results in short, thin tau filaments that are scattered in neuronal somata. In contrast, GFPtagged tau filaments are longer, thicker and more densely packed in clusters distributed in somata, but also in neurites. The GVB load in the untagged tau model was approximately 10x higher than in the tau-GFP model. Our results indicate that aggregate structure and/or subcellular localization are possible determinants of GVB accumulation. To gain a deeper understanding of the effects of tau aggregation in neurons, proteomics analysis of the primary neuron model for tau pathology was performed. The results revealed a downregulation of pathways associated with the lysosomal system, which was further studied in Chapter 4. For morphological characterization of lysosomes in these neurons, LysoFinder was developed: a tool for unbiased and semi-automated analysis of vesicular structures in confocal images. In addition, an indicator of cellular proteolytic capacity was employed. Our data show that neurons with tau aggregates contain less lysosomes and accumulate degraded cargo. Upon grouping neurons based on their GVB status, it was shown that GVB-containing neurons are resilient to these tau aggregation-induced changes, which was accompanied by a small but significant increase in TFE3 activation.