Modelling PTEN loss of function in the adult cerebellum to elucidate mechanisms of cerebellar hamartoma.

PTEN mutations are associated with PTEN hamartoma tumor syndromes (PHTS), including Lhermitte-Duclos disease (LDD), a rare cerebellar disorder characterized by the presence of a slowly progressive unilateral mass in the cerebellar cortex. Histopathological hallmarks in LDD, including neuronal hypertrophy and altered cerebellar cytoarchitecture, have been modeled in mice with conditional PTEN deletion during development. However, due to the lethality of constitutive PTEN biallelic deletion, most studies describing the effects of full PTEN loss in specific cerebellar cell types limit the possibility of modeling human PTEN-dependent syndromes. Additionally, in contrast to reported animal models, LDD typically manifests in the third/fourth decade of life, suggesting the involvement of additional mechanisms, comprising the possibility of a ‘second hit’ mutation that inactivates the wild-type allele of PTEN. In this study, we tested the hypothesis that sporadically full PTEN loss in the adult cerebellum, occurring in a hemizygous environment, contributes to the pathogenesis of cerebellar hamartoma syndrome. To investigate this, we develop a novel mouse model by inducing in the adulthood full PTEN knockout (KO) in a subset of cerebellar cells within a hemizygous environment. Combining immunohistochemistry, 3D segmentation and quantitative morphological analysis of Purkinje cells we demonstrated that the full PTEN loss in adulthood in our model is sufficient to induce morphological alterations of the dendritic compartment, supporting the second hit hypothesis. Notably, these changes occur not only in full PTEN KO Purkinje cells but also in adjacent hemizygous or wild-type neurons, thereby demonstrating PTEN acting both in a cell-autonomous and non-cell-autonomous manner. Moreover, we observed that PTEN hemizygosity alone did not induce significant changes, indicating that full PTEN loss drives the observed phenotype. Remarkably, PTEN deletion does not affect Purkinje cell dendritic complexity and spatial coverage. Moreover, we showed that transient pharmacological PTEN inhibition could not replicate the morphological changes described in genetic PTEN KO conditions, but revealed axonal pathological alterations. This discrepancy suggests that only a complete and irreversible PTEN loss of function can drive dendrite alterations, which transient inhibition fails to replicate. These results highlight the impact of PTEN loss in adulthood on Purkinje cell architecture and highlight its broader impact on surrounding neuronal populations. Notably, the non-cell-autonomous effects observed suggest the involvement of additional signaling pathways that mediate interactions between PTEN-deficient and neighboring neurons. In the future, deeper characterization of the molecular changes in these cells will help elucidate the disease progression mechanisms. Overall this research provides a ground for future research on LDD pathogenesis.