Dottorato in NEUROSCIENZE DEL COMPORTAMENTO

Titolo della tesi: The impact of cholesterol dyshomeostasis on neuronal development and functional maturation: lesson from mouse models of Niemann Pick C disease

The Niemann-Pick Type C Disease (NPCD) is a rare and devastating lysosomal storage disorder (LSD) characterized by impaired Cholesterol (Chol) intracellular trafficking and abnormal accumulation of lipids within cellular compartments. It is an autosomal recessive disorder caused by mutations in the NPC1or NPC2 genes, which encode for proteins involved in the exit of Chol from late endosomes/lysosomes. One hallmark of NPCD pathology is progressive neurodegeneration, which is accompanied by a wide range of neurological symptoms. Emerging evidence indicate that NPCD pathology disrupt neurodevelopmental pathways, impacting neuronal differentiation, proliferation and maturation (Kavetsky et al., 2019; Boyle et al., 2020; Kim et al., 2023). Indeed, in the last decade, our group has described the presence of various anomalies in neuronal differentiation using mouse models of NPCD. Altered neurodevelopment has been linked to the disruptions of Shh signaling at the primary cilium (Nusca et al., 2014; Caporali et al., 2016; Lucarelli et al., 2023). More recently, we reasoned that continuous integration of newly generated neurons within the Olfactory Bulb (OB) provides an intriguing model to further explore these anomalies. In addition, olfactory deficits are a prominent characteristic of various neurodegenerative diseases (Doty, Deems, and Stellar 1988; Djordjevic et al. 2008), including NPCD (Hovakimyan et al. 2013). Based on this premise, in this study we have determined how Npc1 deficiency affects: i) patterns of OB development using Npc1 mouse models; ii) patterns of neural development exploiting in vitro cultured neurospheres obtained from Npc1 mice. Moreover, we have identified the molecular basis of abnormal OB and neuronal development by focusing our attention on Shh and redox signaling pathway. The analysis of OB cytoarchitecture in Npc1 mice has revealed a disarray of normal layering, which is particularly pronounced at the boundary between granule and mitral cell layers. Then, the neurochemical profiling of periglomerular granule neurons (PGN) has shown alterations in the numbers of various PGN subtypes. Specifically, our analysis has shown that the number of Calretinin (Calr)-positive, Calbindin (Calb)-positive and Tyrosine Hydrolase (TH)-positive PGNs in the OB of Npc1 mice is significantly higher, whereas the number of Parvalbumin (Parv)-positive PGNs is significantly lower compared to wt ones, further highlighting the presence of a cytoarchitectural disorganization, which reasonably explains defective olfaction in Npc1 mice (Rava et al., 2022). The analysis of the expression of Shh signaling components has revealed significant differences in Shh, Patched1 (Ptch1) and Smoothened (Smo) transcript levels starting from a very early a-/pre-symptomatic age, such as Post Natal Day (PND) 15. Specifically, PND15 Npc1 mice show increased Shh and Smo transcript levels and a decrease of Ptch1 transcripts, whereas at later ages there is a global decrease of all three factors. This suggests an initial compensatory increase of Shh and Smo, which is then followed by a reduction of their expression as Chol dyshomeostasis worsen with animal aging. Dysregulation of Glis factors, the transcriptional effectors of Shh signaling, further validated Shh pathway disruption in the OB of Npc1mice. Accordingly, in this study we have also found that Npc1 mice display a significant reduction in odorant receptor (OR) expression and a decline in S100A5 – an early marker of OR activation. Given that the disruption in Shh signaling causes the dysregulation of cellular processes, including cell proliferation, differentiation, and survival (Martínez et al. 2020; Yabut et al. 2020), we next addressed our interest to the analysis of these neurodevelopmental processes. In vitro studies on Npc1-/- Neural Stem Cells (NSCs) have shown an imbalance between proliferation and differentiation. Specifically, Npc1-/- NSCs display a reduced self-renewal capacity and a major degree of differentiation towards the neuronal lineage compared to wt ones. These findings have been strengthened by the analysis of developing cortical neural precursors in Npc1-/- mice, which disclosed a similar imbalance between proliferation and differentiation. Indeed, we have observed a decreased proliferation of Radial Glial Cells (RGCs) associated to an increased number of Intermediate Progenitor Cells (IPCs) and differentiated neurons. To gain an insight on molecular mechanisms responsible for abnormal neuronal differentiation, we have addressed our interest to mitochondria and redox signaling because they have recently emerged as a key component of NPCD neuropathology. Specifically, we have investigated mitochondrial metabolic and scavenger activities, and ROS buffering capacity in Murine Embryonic Fibroblasts (MEFs) obtained from wt and Npc1-/- mice. By this analysis we have demonstrated the presence of decreased OXPHOS and elevated reactive oxygen species (ROS) levels in Npc1-/- MEFs compared to wt ones. Our focus on redox signaling regulation, with particular reference to the analysis of nuclear factor erythroid 2–related factor 2 (Nrf2), has shown the lack of Nrf2 nuclear translocation in Npc1-/- MEFs. This, in turn hinders the antioxidant response, as indicated by the decreased expression of typical antioxidant genes, including Nqo1, Gcl, Gpx4. Furthermore, we have demonstrated there is a considerable drop in the Gpx4 protein content, which is essential for reducing Oxidative Stress (OS). Collectively, these results indicate that Npc1 cells have a compromised antioxidant response. In conclusion, this multiapproach study has provided relevant insights into the pathophysiology of NPCD, laying the groundwork for potential therapeutic interventions targeting the cellular/molecular processes that we found to be altered.