A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition

Angireddy, R.; Murali, S.; Karisetty, B. C.; Kossenkov, A.; Katsura, K.; Ohl, L.; Smith, S.; Clark, K.; Bhoj, E.; Diaz, A.

doi:10.1101/2024.10.30.621078

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bioRxiv · 10.1101/2024.10.30.621078

A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition

Angireddy, R.·Murali, S.·Karisetty, B. C.·Kossenkov, A.·Katsura, K.·Ohl, L.·Smith, S.·Clark, K.·Bhoj, E.·Diaz, A.

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Abstract

Background and ObjectivesTBCK syndrome is a rare fatal pediatric neurodegenerative disease caused by biallelic loss-of-function mutations in the TBCK gene. Previous studies by our lab and others have implicated mTOR, autophagy, lysosomes, and intracellular mRNA transport, however the exact primary pathologic mechanism is unknown. This gap has prevented the development of targeted therapies. MethodsWe employed a human neural progenitor cell line (NPC), ReNcell VM, which can differentiate into neurons and astrocytes, to understand the role of TBCK in mTORC1 activity and neuronal autophagy and cellular mechanisms of pathology. We used shRNA technology to knockdown TBCK in ReNcells. ResultsThese data showed that loss of TBCK did not inhibit mTORC1 activity in neither NPC nor neurons. Additionally, analysis of eight patient-derived cells and TBCK knock down HeLa cells showed that mTORC1 inhibition is inconsistent across different patients and cell types. We showed that TBCK knockdown in ReNcells affected NPC differentiation to neurons and astrocytes. Specifically, differentiation defects are coupled to cell cycle defects in NPC and increased cell death during differentiation. RNAseq analysis indicated the downregulation of several different neurodevelopmental and differentiation pathways. We observed a higher number of LC3-positive vesicles in the soma and neurites of TBCK knockdown cells. Further, TBCK knockdown altered mitochondrial dynamics and membrane potential in NPC, neurons and astrocytes. We found partial mitochondrial rescue with the mitochondrial fission inhibitor mdivi- 1. DiscussionThis work outlines a new Human Cell Model for TBCK-related neurodegeneration and the essential role of mitochondrial health and partial rescue with mitochondrial fission inhibitor. This data, along with human neurons and astrocytes, illuminate mechanisms of neurodegeneration and provide a possible novel therapeutic avenue for affected patients.

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Angireddy, R., Murali, S., Karisetty, B. C., Kossenkov, A., Katsura, K., Ohl, L., Smith, S., Clark, K., Bhoj, E., Diaz, A.. 2024-10-31. A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition. https://doi.org/10.1101/2024.10.30.621078

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