Abate, Natalia (2025) Unveiling CSTB-dependent molecular and cellular alterations in neuronal development in EPM1: from protein synthesis to extracellular vesicles secretion. [Tesi di dottorato]

Documento PDF PhDthesis_NataliaAbate.pdf Download (2MB)

Abstract

Unverricht–Lundborg disease (EPM1) is the most common form of progressive myoclonic epilepsy, caused by loss-of-function mutations in the CSTB gene. Beyond its established role as a cysteine protease inhibitor, CSTB participates in key neurodevelopmental processes, including neuronal proliferation, differentiation and synaptic plasticity. To elucidate CSTB functions and uncover mechanisms underlying EPM1 pathogenesis, we employed human iPSC-derived cerebral organoids with dorsal and ventral cortical identities to drive cell fate toward excitatory or inhibitory neuronal differentiation respectively. At advanced maturation stages of cerebral organoids, co-immunoprecipitation and mass spectrometry analyses revealed that CSTB specifically interacts with the CCT/TRiC chaperonin complex, a regulator of cytoskeletal protein folding, translation activity and vesicle biogenesis. This interaction is specific of ventrally patterned cerebral organoids, therefore we investigated CCT-dependent processes in two experimental models derived from EPM1 iPSCs: i) ventral neural progenitor cells (vNPCs) and ii) vNPCs induced to neuronal differentiation for three days (vNPCs-D3). Our results demonstrate that cellular alterations emerge immediately upon neuronal differentiation in EPM1 cells. Protein synthesis becomes impaired at very early stages of neuronal induction and persists throughout neuronal maturation. At early stages, EPM1 cells also exhibit fragmented actin filaments and perinuclear accumulation of late endosome markers, indicating defective maturation of multivesicular bodies and impaired secretion of extracellular vesicles (EVs). We also reproduced EPM1 defects by silencing TCP-1, a core CCT subunit, in control neurons supporting the functional relevance of CTSB-CCT interaction. Moreover, proteomic analysis of EVs released by EPM1 neurons during maturation revealed depletion of proteins involved in cytoskeletal organization, synaptic vesicle trafficking and calcium-dependent exocytosis. We also investigated CSTB secretion via EVs using adult rat brain synaptosomes as model system and demonstrated that CSTB secretion is depolarization-dependent and regulated by extracellular and intracellular calcium balance, which also influences the secretion of CD81-positive EVs subpopulations. Overall, this work shows that EPM1-related cellular alterations, mediated by pathological missing interaction between CSTB and CCT complex, arise at the earliest stages of neuronal differentiation. The data highlight a key role for CSTB–CCT cooperation in coordinating cytoskeletal remodeling, vesicle-mediated signaling and synaptic homeostasis, providing novel insights into EPM1 pathophysiology and potential targets for therapeutic intervention.

Downloads

Actions (login required)

Modifica documento