Abstract
Overview: A role for p53 isoforms in largely promoting cancer has been established. One isoform, ∆133p53, promotes numerous aspects of cancer namely inflammation, proliferation and invasion. Δ133p53 functions are also involved in reducing cell death while promoting survival, leading to brain tumours and other diseases in the brain, such as neurodegeneration. A mouse model that mimics ∆133p53 function, ∆122p53, has aided the understanding of ∆133p53 function. This study explores a role for ∆133p53 in neuropathology which was investigated using two ∆122p53 models: the original ∆122p53 model where ∆122p53 was present in every cell type and a new model, MOGLI, where ∆122p53 expression was restricted to glial cells.
Hypothesis: If Δ133p53 is involved in neurodegeneration, histological assessment of the original Δ122p53 mice and MOGLI mice will show neuropathological evidence.
Methods: Mice cohorts were aged and euthanized on showing symptoms such as seizures or major loss in body weight, or after a decided time had passed. Brains were harvested and processed into formalin fixed paraffin embedded (FFPE) blocks. Brains of the original ∆122p53 mice, where ∆122p53 is expressed in every cell type, were investigated for neuropathology using routine histology staining (Haematoxylin and Eosin (H&E)), special staining (iron deposition using Perls Prussian blue), and immunohistochemistry (glial cell glial fibrillary acidic protein, GFAP), T lymphocyte (CD3) and M2 macrophage/activated microglia (CD163) markers. Mice heterozygote for full-length p53 and a p53 null allele (p53+/-) were used as controls. The survival of MOGLI model, where ∆122p53 was restricted to glial cells, was investigated. Brain tissue from the MOGLI mice (GCE ∆122p53+/+ and GCE ∆122/p53+) and its controls (GCE m∆pro+/+, GCE m∆pro+/-, m∆pro+/- and GCE wild type+/+ mice) were also investigated for neuropathology as the original model with additional special staining for myelin loss (Luxol fast blue staining), and in situ hybridisation analyses for proinflammatory cytokine expression (IL-6 and IFN) using RNAscope®. Lastly, this study also aimed at analysing protein cell surface expression data from Δ133p53 expressing monoclonal cell lines to check if any proteins implicated in neurodegenerative pathology are increased or decreased on the Δ133p53 cell surface compared to parent cell line (H1299: A human non-small cell lung carcinoma cell line), which acted as the control. Statistical analysis on all the relevant data obtained throughout the study was performed using Prism 9 software.
Results: The original ∆122p53 mouse model, which had not shown any physical neurological symptoms, had neuropathology on analysis of H&E staining with the presence of a greater number of microcysts compared to the control mice (p = 0.0014). Immunohistochemistry and iron staining did not reveal the underlying cause of the microcysts nor display any evidence for another type of neuropathology. The MOGLI test mice developed an array of neurological symptoms, suggesting glioblastoma development. Of the 43 MOGLI test mice, 42 mice displayed at least 1 neurological symptom or adverse effect that required euthanasia, while none of the 11 control mice displayed any signs of neurological distress. MOGLI mice had reduced survival compared to the control mice (p < 0.0001). A sex difference in overall survival was found for MOGLI test mice, in which male mice heterozygote and homozygote for
∆122p53 had a reduced median survival time compared to female homozygote (p = 0.0386) and heterozygote (p = 0.00178) MOGLI mice. Haematoxylin and Eosin staining showed that glioblastomas or other tumours were not present in the MOGLI mice. Instead MOGLI mice had other features of neuropathology i.e., foamy macrophage cells (8/43, 18%), mononuclear aggregates (5/43, 12%) and hypercellularity (13/43, 30%). These features were absent in control animals. An unknown brown pigment was also seen in some MOGLI test (19/43, 44%) as well as control mice (6/11, 55%). Immunohistochemistry, iron staining, myelin staining and RNAscope® were not able to clarify the underlying cause or characterise further the above neuropathologies.
Cell surface protein expression analysis of Δ133p53 expressing cell lines revealed 34 upregulated proteins and 32 downregulated proteins in Δ133p53 expressing cells compared to the control. Of the altered proteins, 10 upregulated and 9 downregulated proteins were myelin sheath proteins. Among the upregulated proteins, clathrin (CLTC) showed highest expression and among the downregulated proteins CCT2 had no expression at all.
Conclusion: This study identified neuropathologies in the original Δ122p53 and the MOGLI mouse model, but did not find the underlying cause of the pathologies. Nonetheless, it revealed some new avenues to explore. It showed the presence of microcysts in the ∆122p53 mouse model which have not been reported previously. Foamy macrophages, a characteristic of lipid storage diseases, were seen in some MOGLI mice. Inflammatory hypercellularity and mononuclear aggregates were seen in some MOGLI mice, but were never observed occurring together. An unidentified brown pigment, of unknown significance, was seen in some of the MOGLI test as well as control mice, with varying quantity distribution across the genotypes and sexes. Difference in survival times was also observed between the MOGLI test and control mice, with significant variations between sexes for the MOGLI test mice. Lastly, protein expression analysis of Δ133p53 expressing cell lines revealed upregulation of proteins involved in vesicle transport and molecular trafficking.