Nucleolin reorganization and nucleolar stress in Purkinje cells of mutant PCD mice

The Purkinje cell (PC) degeneration (pcd) mouse harbors a mutation in Agtpbp1 gene that encodes for the cytosolic carboxypeptidase, CCP1. The mutation causes degeneration and death of PCs during the postnatal life, resulting in clinical and pathological manifestation of cerebellar ataxia. Monogenic biallelic damaging variants in the Agtpbp1 gene cause infantile-onset neurodegeneration and cerebellar atrophy, linking loss of functional CCP1 with human neurodegeneration. Although CCP1 plays a key role in the regulation of tubulin stabilization, its loss of function in PCs leads to a severe nuclear phenotype with heterochromatinization and accumulation of DNA damage. Therefore, the pcd mice provides a useful neuronal model to investigate nuclear mechanisms involved in neurodegeneration, particularly the nucleolar stress. In this study, we demonstrated that the Agtpbp1 gene mutation induces a p53-dependent nucleolar stress response in PCs, which is characterized by nucleolar fragmentation, nucleoplasmic and cytoplasmic mislocalization of nucleolin, and dysfunction of both pre-rRNA processing and mRNA translation. RT-qPCR analysis revealed reduction of mature 18S rRNA, with a parallel increase of its intermediate 18S-5'-ETS precursor, that correlates with a reduced expression of Fbl mRNA, which encodes an essential factor for rRNA processing. Moreover, nucleolar alterations were accompanied by a reduction of PTEN mRNA and protein levels, which appears to be related to the chromosome instability and accumulation of DNA damage in degenerating PCs. Our results highlight the essential contribution of nucleolar stress to PC degeneration and also underscore the nucleoplasmic mislocalization of nucleolin as a potential indicator of neurodegenerative processes.