Proteostasis mechanisms in direct neuronal reprogramming

Malpartida, Ana Belen (2026): Proteostasis mechanisms in direct neuronal reprogramming. Dissertation, LMU München: Faculty of Medicine

Abstract

Despite significant progress in the field of direct neuronal reprogramming, there are still many challenges that need to be addressed prior to clinical application. A deeper understanding of the mechanisms driving fate conversion, as well as the specific hurdles that hinder this process, is essential to optimize conversion efficiency and enable the application of neuronal reprogramming as a therapeutic strategy. The focus of my PhD thesis was to address these hurdles and determine their impact on direct neuronal reprogramming focusing on mitochondrial dysfunction, impairments in proteostasis and protein aggregation. Overall, I aimed to understand the involvement of different proteostasis mechanisms during direct neuronal reprogramming, when many new proteins have to be made to install the new cell fate and others have to be degraded to abolish the glia-specific proteins. This is relevant for reprogramming of healthy cells, but even more so in neurodegenerative diseases with protein aggregate loads to probe and further develop this approach as a viable therapeutic intervention in chronic disease. Following the finding that mitochondrial dysfunction poses a major hurdle in mouse astrocyte to neuron conversion, we determined the impact of mitochondrial dysfunction in the reprogramming of human cells by generating proliferating astrocytes from patient iPSCs carrying mutations in the NDUFS4 gene as a model for Complex I dysfunction. Pharmacological targeting of distinct aspects of mitochondrial function and ER stress allowed us to identify the unfolded protein response (UPR) as a novel, general hurdle in direct neuronal reprogramming. Specifically, we propose ER-stress mediated inhibition of translation by the PERK pathway as a critical hurdle in the early stages of neuronal conversion. Importantly, we demonstrated that pharmacological inhibition of the UPR significantly improves the neuronal conversion of both control and patient astrocytes, suggesting its potential to facilitate neuronal conversion in health and disease. Moreover, we determined that protein synthesis levels increase during direct neuronal reprogramming, leading to an accumulation of misfolded proteins and eventual activation of the UPR. Altogether, this work highlights the key importance of proteostasis during early stages of neuronal conversion and suggests that processes like translation, protein folding and UPR activation act as major hurdles in the neuronal reprogramming of human cells. Given the implication of proteostasis mechanisms in direct neuronal reprogramming, it then became crucial to explore this strategy in a disease model where protein homeostasis is disrupted, as is the case in most neurodegenerative diseases. Using a mouse model of ALS recapitulating C9orf72 pathology, we determined that protein aggregation and impaired proteasome function significantly decrease neuronal conversion. While we propose strategies to overcome these limitations, their success may be limited by the stage of disease progression. Pharmacological modulation of proteasomal activity resulted in only mild improvements in the reprogramming of transgenic astrocytes, with aggregate load remaining unchanged. Notably, promoting protein degradation to prevent protein misfolding in healthy human astrocytes did not improve reprogramming efficiency, suggesting a complex interplay between protein quality control mechanisms and cell fate conversion. In summary, my PhD aimed to unravel key hurdles in direct neuronal reprogramming, particularly those arising from chronic disease states. This thesis proposes proteostasis mechanisms as a bottleneck for direct neuronal reprogramming and provides insights into how current strategies could be optimized to apply direct neuronal reprogramming as a viable therapeutic strategy for chronic and neurodegenerative diseases.

• 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: 10. March 2026
• 1. Referee: Götz, Magdalena
• Signature of the printed copy: 0700/UMD 22777