Ubiquitination is one of the most abundant and versatile post-translation modifications in eukaryotes that affects many biological processes by modifying protein activity, interactions, localization and stability of substrates. E3 ligases have a key function in the process, acting as molecular ubiquitin-substrate matchmakers and providing specificity to the reaction. In this thesis, we aimed at characterizing the physiological and pathological functions of the human HECW1, a poorly studied E3 ligase which belongs to the NEDD4 family. HECW1 is preferentially expressed in the central nervous system (CNS) and it has been linked to neurodegeneration, in particular to the familial form of Amyotrophic Lateral Sclerosis (fALS). The Drosophila orthologue Hecw, that we recently identified and functionally characterized, is similarly enriched in the CNS and is involved in the dynamic regulation of RNPs required for neuronal health. The Hecw/HECW1 interactome is enriched in proteins involved in the autophagy/endolysosomal pathway and RNPs dynamics, whose dysfunction promote neurodegenerative diseases. Together these data suggest a protective role of HECW1 in neuronal homeostasis. To investigate into HECW1 physiological and disease-relevant neuronal function, we optimized a protocol to directly differentiate neurons from human iPSCs and we generated HECW1-KO iPSCs. We found that HECW1 expression in neurons is upregulated during differentiation and downregulated with aging, a typical behavior of components of the ubiquitin proteasome and autophagy pathways. Unbiased proteomic analysis showed deregulation of proteins involved in vesicle traffic and kinase activity in HECW-KO neurons. Targeted immunofluorescence, morphological and EM analysis revealed an accumulation of enlarged organelles positive for the autophagic and endo-lysosomal marker LAMP1 and of endolysosomal/autophagic compartments structures along filaments and in distal axons of HECW1-depleted neurons. Moreover, distal tips of HECW1-KO neurons showed the accumulation of abnormal, static WGA-aggregates, indicating an impairment in endosomal traffic. A second category of enriched HECW1 interactors is related to RNA metabolism. Co-immunoprecipitation analysis confirmed HECW1 interaction with the SG protein FMRP and the PB component EDC3. We also measured an increase number of constitutive PBs in HECW1-depleted neurons, suggesting a possible regulatory role of HECW1 in constitutive PB formation or clearance. The two phenotypes observed in HECW1-depleted neurons could be functionally linked, considering the involvement of autophagy in the clearance of persistent RNPs arising from chronic stress or disease mutations. Taken together, our results have uncovered an involvement of HECW1 in the regulation of the autophagy/endolysosomal pathway in neurons and a possible contribution in controlling the homeostasis of ribonucleoprotein particles. Future studies in motor neurons would help to understand the implications of these results for ASL, a disease where dysregulation of RNA metabolism, cytoplasmic mislocalization of RNA binding proteins and dysfunction in RNP dynamics, along with autophagy impairments appear to be at the basis of the pathogenesis.
THE E3 UBIQUITIN LIGASE HECW1 IN NEURONAL HOMEOSTASIS / S. Sala ; internal SEMM advisor: E. Dejana ; external SEMM advisor: Y. Kulathu ; coordinator: S. Minucci ; tutor: S. Polo. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2021 Dec 13. ((33. ciclo, Anno Accademico 2021.
|Titolo:||THE E3 UBIQUITIN LIGASE HECW1 IN NEURONAL HOMEOSTASIS|
|Supervisori e coordinatori interni:||MINUCCI, SAVERIO|
|Data di pubblicazione:||13-dic-2021|
|Parole Chiave:||ubiquitin; HECW1; E3 ligase; iPSC; human neurons; neurodegeneration; autophagy; endolysosomal pathway; ribonucleoprotein particles RNPs|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||THE E3 UBIQUITIN LIGASE HECW1 IN NEURONAL HOMEOSTASIS / S. Sala ; internal SEMM advisor: E. Dejana ; external SEMM advisor: Y. Kulathu ; coordinator: S. Minucci ; tutor: S. Polo. - Milano : Università degli studi di Milano. Università degli Studi di Milano, 2021 Dec 13. ((33. ciclo, Anno Accademico 2021.|
|Appare nelle tipologie:||Tesi di dottorato|