Lysosomal dysfunction and microglial hyperactivation in models of progranulin deficiency

Reifschneider, Anika (2023): Lysosomal dysfunction and microglial hyperactivation in models of progranulin deficiency. Dissertation, LMU München: Medizinische Fakultät

Abstract

In my thesis I focused on the pivotal role of microglia in neurodegenerative disease, their different activation stages upon progranulin (PGRN) or triggering receptor expressed on myeloid cells 2 (TREM2) deficiency and the connection between lysosomal deficiency and microglial hyperactivation. Microglia majorly contribute to the progression and pathology of neurodegenerative disorders like Alzheimer`s disease (AD) and frontotemporal lobal degeneration (FTLD) and additionally recent advances of genome wide association studies (GWAS) have identified genetic association, as rare variants of genes that are predominantly expressed by microglia increase the risk of developing neurodegenerative disease. Among these risk genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). While the heterozygous loss of PGRN leads to FTLD, the complete loss of PGRN results in the lysosomal storage disease neuronal ceroid lipofuscinosis, indicating a major role of PGRN in lysosomal protein degradation in the brain. Our laboratory has previously shown, that progranulin knockout mice (Grn-/-) and GRN-associated FTLD patients exhibit increased levels of the lysosomal protein cathepsin (Cat) D, however the exact role of PGRN in lysosomal protein degradation remained unclear. In a collaborative effort with Julia K. Götzl, Alessio-Vittorio Colombo and Kathrin Fellerer, I therefore analyzed microglia and other brain cells regarding changes in expression, maturation and enzymatic activity of lysosomal proteins like Cat D, B and L. We found a striking age-depended increase of lysosomal proteases associated with increased enzymatic activity. Interestingly, we demonstrated that microglia show early lysosomal deficits, even before enhanced Cat transcription levels were observed. Our laboratory has previously shown, that PGRN loss of function (LOF) leads to hyperactivated microglia that exhibit increased phagocytosis, proliferation and migration. The opposite microglial phenotype is found in TREM2 LOF models, where microglia appear to be locked in a homeostatic state, unable to react to pathological insults. In addition to the lysosomal dysfunction discussed above, PGRN LOF microglia also increase TREM2 expression. To test the hypothesis that hyperactivation of microglia in PGRN LOF is TREM2-dependent and that microglia can reversibly switch between activation stages, I used genetic and pharmacological TREM2 antagonistic approaches to prevent the transition of homeostatic microglia to a disease-associated microglia (DAM) state. To further investigate the microglial contribution to disease pathology in PGRN LOF models, I generated Grn x Trem2 double knockout mice to analyze the expression of DAM genes, lysosomal dysfunction, glucose uptake, lipid metabolism and microglia morphology and activation status. Here, I found that ablating TREM2 in PGRN LOF mice reduces the expression of DAM genes, suggesting that suppression of TREM2 can lower microglia hyperactivation and is likely to be upstream PGRN-mediated microglial transcriptional changes. To further explore whether pharmacological modulation of TREM2 has beneficial functions on microglia states, I used antibodies antagonistic for TREM2, developed at Denali Therapeutics, to treat macrophages isolated from GRN-FTLD patients. Treatment of the cells with these antibodies resulted in reduced TREM2 signaling, due to its enhanced shedding. To confirm these findings, I collaborated with Sophie Robinson, who generated PGRN-deficient microglia derived from human-induced pluripotent stem cells (iPSC). Treatment of these cells with antagonistic TREM2 antibodies resulted in reduced microglia hyperactivation, TREM2 signaling and phagocytic activity. However, we did not observe any effects on lysosomal dysfunction in PGRN deficient iPSC after antibody treatment. In line with this, Grn x Trem2 double knockout mice not only failed to rescue effects on lysosomal dysfunction, lipid metabolism and microglia morphology, but also further increased synaptic loss and neurofilament light-chain (Nfl) levels, a marker of neuronal damage in the brain. My results suggest that with PGRN deficiency, lysosomal dysfunction is upstream to the microglia hyperactivation. In addition, these findings imply a protective role of TREM2-dependent chronic activation of microglia and show the dynamic nature of microglia kinetics and their ability to reversibly switch between activation stages.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: microglia, progranulin, lysosomes, neurodegeneration, frontotemporal lobal degeneration
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Februar 2023
• 1. Bericht­erstatter:in: Haass, Christian