Oxidative stress and neuroinflammation effects in hippocampal development, aging, and disease

This PhD thesis aims to evaluate oxidative stress and inflammation pathways considering three pathological models such as Prolidase Deficiency, Physiological Aging and Anorexia Nervosa. Furthermore, preliminary experiments were performed to better understand the involvement of different cell death pathways i.e. apoptosis, autophagy and mitophagy. Prolidase deficiency (PD) is a rare genetic disorder caused by a mutation in the PEPD gene that codes for the prolidase enzyme. This enzyme can selectively cut proline dimers present at the carboxy-terminal end of a polypeptide and participates in the turnover of collagen. Recently published data have demonstrated the presence of cerebellar morphological alterations in PD mice, extracellular matrix disorganization, and consequently tissue damage and anomalies in cell migration [1]. Ageing is a condition closely related to oxidative stress and inflammation that physiologically affects everyone. Recently, it has been shown that the medicinal mushroom Hericium erinaceus (He1) has antioxidant activity and partially reactivates cell proliferation in some brain areas of physiologically aged mice and animals subjected to a diet supplemented with the mushroom [2] Eating Disorders are common, complex, and difficult-to-cure diseases that mostly arise during adolescence. Among the various disorders, Anorexia Nervosa (AN) was considered a pathology characterized by an insufficient energy supply for survival, with consequences that affect the whole organism and affect endocrine, cardiovascular, gastrointestinal, haematological, and skeletal muscle functions. Oxidative stress and consequently inflammation seem to be a common thread in these three pathological models and could trigger several types of cell death. Morphological and immunohistochemical reactions were conducted on mouse and human hippocampal sections considering the following antibodies: superoxide dismutase 1 and 2 (SOD1, SOD2), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2), interleukin 6 (IL-6), and transforming growth factor β (TGF-β). In addition, for the cell death pathways Bax, Bcl2, p62, PINK1 and PARKIN were evaluated. Haematoxylin and eosin staining, and Nissl staining were also performed for cytoarchitectural analyses. The purpose of this thesis was to determine differences in the expression of antioxidant proteins, inflammatory cytokines and molecules and cell death markers in the hippocampal formation of mice affected by PD (dal) at 10 days after birth (P10), P21 and P60, aged mice supplemented for 2 months with 1 mg/day of standardized extracts of He1 and human brain biopsies of a control and AN patient. The results were obtained by using microtome cutting techniques, immunohistochemistry and immunofluorescence experiments and optical microscope analysis followed by statistical evaluations. First, the morphological differences in the dentate gyrus of the hippocampus were evaluated by Haematoxylin/eosin and Nissl staining and further the expression of specific markers for oxidative stress and inflammation, i.e. SOD1, SOD2, COX4, NOS2, COX2, IL- 6, TGF-β and cell death phenomena due to apoptosis, i.e. Bcl-2 and Bax, autophagy, i.e. p62 and mitophagy, i.e. PINK1 and PARKIN were evaluated. From these analyses, it was possible to observe how a strong oxidative stress and inflammation environment is present in pathological conditions and contributes to activate mechanisms of regulated cell death. Most of the observed cells expressed markers of autophagy and mitophagy rather than apoptosis, a sign, however, of an attempt by the cells to eliminate the harmful components without causing further damage to the tissue. In conclusion, we can therefore think that these data can provide insights to improve an antioxidative and antiinflammation treatment through activation of programmed cell death for cell survival.