Modulation of lysosomal proteases in FTD-GRN

Mühlhofer, Quirin (2026): Modulation of lysosomal proteases in FTD-GRN. Dissertation, LMU München: Faculty of Medicine

Abstract

One of the most common genetic risk factors associated with frontotemporal lobar degeneration (FTLD) is GRN haploinsufficiency. However, inclusions of TAR-DNA binding protein 43 (TDP-43) are the major pathological hallmark of progranulin (PGRN) related FTLD (Grn-FTLD). Recently, the lysosomal protease legumain (LGMN) has been proposed to mechanistically link the loss of PGRN to TDP-43 pathology in FTLD. LGMN is capable of generating pathological TDP-43 fragments and upregulation of LGMN activity has been detected in brains of GRN-FTLD patients with TDP-43 pathology. Besides TDP-43, several proteins involved in various neurodegenerative diseases such as tau, amyloid precursor protein (APP) and α-synuclein are also cleaved by LGMN. Hence, modulation of LGMN activity turned out to be a promising new therapeutic target. This work provides two different approaches for the modulation of LGMN activity. One strategy was to analyze the effect of the endogenous cysteine protease inhibitors cystatin-3 and cystatin-7 on maturation and activity of LGMN. The other approach focused on restoring PGRN, a protein which has been shown to dampen LGMN maturation and proteolytic activity. In addition to in vitro studies, the effects of PGRN, cystatin-3 and cystatin-7 were analyzed in the mouse brain, an environment resembling the situation in the human brain. Therefore, transgene expression of the three proteins was induced by ultrasound-guided intraventricular adeno-associated virus (AAV) injection into neonatal mice. In this thesis, an AAV9 capsid containing the respective gene of interest was used. 3 months post injection, strong and widespread protein expression was detected. Areas around the lateral ventricles exhibited the highest expression and the hSyn promoter enabled pronounced expression in neurons. Cystatin-3 as well as cystatin-7 reduced LGMN activity in vitro and in vivo. Incubation of the respective recombinant inhibitors and LGMN revealed that cystatin-7 delayed maturation of LGMN much more efficiently than cystatin-3. For half-maximal inhibition of active LGMN lower concentrations of cystatin-7 were needed whereas cystatin-3 appeared to be better suited for complete LGMN inhibition. In contrast to the mainly unaffected cystatin-3 treated mice, cystatin-7 overexpressing mice showed enlarged lysosomes and minor signs of gliosis combined with compromised motor skills. To assess further effects of a cystatin treatment, the influence of both cystatins on other lysosomal proteases, in particular cathepsins was analyzed. Overexpression of cystatin-3 and -7 in HEK 293T cells and C57BL/6J mouse brains differentially changed the activity and maturation of cathepsins D, B and L next to LGMN. In summary, cystatin-3 appeared as a more favorable candidate for future studies with the aim of modulating LGMN activity. Since GRN-FTLD is caused by GRN haploinsufficiency, boosting PGRN expression seems a logical treatment strategy. Various studies support this idea with positive results obtained from expressing PGRN in Grn knockout mice. In this study, Grn-/-/Tmem106b-/- mice, a model for GRN-FTLD with accelerated pathology concerning lysosomal function, proteostasis, gliosis and motor function, were used for AAV-mediated PGRN delivery. At an age of 3 months, untreated Grn-/-/Tmem106b-/- mice exhibited pathological hallmarks including microgliosis, astrogliosis, impaired autophagy, increased activity of lysosomal proteases and beginning TDP-43-like pathology. Intraventricular AAV-Grn injection in neonatal Grn-/-/Tmem106b-/- mice could not rescue pathology but exacerbated motor dysfunction. Furthermore, overexpression of PGRN in wild type mice induced gliosis, impaired autophagy and lead to the upregulation of lysosomal proteases. Thinning of the cortex dorsal of the injection site was accompanied by severely impaired motor function. Therefore, my findings emphasize the importance of further studies on the dose-dependency and the therapeutic window of a PGRN treatment. In conclusion, modulation of LGMN activity as a strategy for the treatment of GRN-FTLD with TDP-43 pathology is worth further research. Different approaches such as boosting endogenous proteins like cystatin-3, cystatin-7 and PGRN, respectively, or the use of small-molecule inhibitors highly specific for LGMN should be compared to determine a powerful method of treatment for this devastating disease.

• Item Type: Theses (Dissertation, LMU Munich)
• Subjects: 600 Technology, Medicine; 600 Technology, Medicine > 610 Medical sciences and medicine
• Faculties: Faculty of Medicine
• Language: English
• Date of oral examination: 5. March 2026
• 1. Referee: Haass, Christian
• Signature of the printed copy: 0700/UMD 22745