Dottorato in NEUROSCIENZE DEL COMPORTAMENTO

Titolo della tesi: Altered BDNF signaling affects postnatal cerebellar development and function in a mouse model of Niemann-Pick type C1 disease

Niemann- Pick type C1 (NPC1) disease is a heterogeneous lysosomal storage disorder due to mutations in the NPC1 gene, encoding a transmembrane protein that mediates the mobilization of cholesterol from endosomes and lysosomes. This mutation causes a misfolding of NPC1 protein, which leads to unesterified cholesterol accumulation within endosomal/lysosomal compartments. The prominent feature of this disorder is the progressive Purkinje cells (PCs) degeneration leading to ataxia. Although NPC1 disease has been considered for long time a neurodegenerative disease, recent studies conducted in our laboratory have demonstrated an abnormal cerebellar development at very early stages of postnatal life in Npc1 mouse models. We demonstrated that the availability and reception of Sonic hedgehog, a mitogenic factor secreted by PCs, is reduced in Npc1 mutant mice, leading to a diminished proliferation of cerebellar granule cells (GCs) in the external granular layer, resulting in an overall reduction in cerebellar size and thickness in adulthood. GCs, representing the largest neuronal population in the brain, have a central role in cerebellar function via their synaptic interactions with other neuronal cell types both within and outside this structure. The development of these cells is based on a timely and precisely orchestrated sequence of proliferation and radial migration from the External granular layer to the Internal granular layer , where they complete differentiation and form synaptic glomerular structures with excitatory mossy fiber axons and inhibitory Golgi cell axons. The precise transition between the different stages of GCs development is Brain-derived neurotrophic factor (BNDF)-regulated and they are essential phenomena for normal development and synaptogenesis, and alterations in the proper differentiative program during this critical period have been linked to major neurodevelopmental disorders. Recently, we observed a significant reduction in BDNF expression levels at very early stages of cerebellar development, in a hypomorphic Npc1nmf164 mouse model that mimics the late-onset and more slowly progressing forms of NPC disease that comprise most human cases. Since pleiotropic effects of BDNF depend on its binding to the tropomyosin kinase B (TrKB) receptor, leading to its autophosphorylation (pTrkB), the current project focused on characterizing the alterations of BDNF-TrkB signaling present in this model mutant Npc1nmf164, which in turn affect GC maturation and synaptogenesis, by altering the cytoarchitecture of the adult cerebellum. During the postnatal cerebellar development of Npc1nmf164 mice, our results show: (i) reduced chemotactic response to BDNF in Npc1 GCs cultured in vitro; (ii) reduced expression of pTrkB in the first weeks postpartum, phases in which GCs complete their proliferation and migration and begin the differentiation into the Internal granular layer; (iii) impaired internalization of the activated TrkB receptor associated with its altered subcellular localization, both in vivo and in vitro; (iv) overall increase in dendritic branching in mature GCs, resulting in compromised differentiation of the glomeruli, the main synaptic area between GCs and Mossy fibers; (v) decreased levels of the SNARE protein, SNAP25; (vi) specific social behavioral anomalies observed only in adult Npc1 male mice, before the onset of neurodegenerative symptoms. Here we propose that impaired BDNF signaling and socially related behavioral deficits in mutant mice are part of complex abnormal developmental trajectories, antecedent to subsequent pathological events associated with NPC1 disease.