Impaired Neuronal Plasticity in Alzheimer's Disease: The Role of Calcium, Insulin and Cell Surface Proteins

Abstract

Neural cell adhesion molecule (NCAM) undergoes post-translational modification by addition of polysialic acid (PSA), which disrupts cell adhesive properties and promotes cell migration, axonal growth, and synaptogenesis. It has been proposed that the decline in memory and cognitive function in Alzheimer’s Disease (AD), is due in-part to a decrease in PSA-NCAM expression in the entorhinal cortex (EC); an area of the brain involved in memory formation, and one of the earliest brain regions affected in AD. In this project, I aimed to examine changes in genes involved in PSA-NCAM regulation through the utilisation of both normal and AD human brain tissue and in vitro neuronal cultures. Initially, a platform was developed allowing the study of mRNA and protein expression on the same tissue section, thus maintaining anatomical context. The approach consisted of RNAscope® followed by immunohistochemistry. This was then applied to investigate the expression of genes involved in the synthesis, cleavage, and recycling of PSA-NCAM in the AD middle temporal gyrus. This revealed a decrease in the expression of PST and STX, polysialyltransferases which synthesize PSA onto NCAM. Next, spatial transcriptomics was conducted to study the expression of twelve genes involved in PSA-NCAM regulation on the same tissue section of EC from AD cases. The results confirmed a decrease in PST high expressing cell population, and a decrease in a CALB2 high expressing cell population, a gene encoding the calcium (Ca²⁺) binding protein calretinin. Finally, I investigated whether Ca²⁺-binding protein overexpression affects PSA-NCAM regulation in vitro. Initially, a differentiation protocol was performed on P19 cells, but transduction efficiencies were very low for Ca²⁺-binding protein expression, so the focus shifted in favour of developing a successful neuronal differentiation protocol for SH-SY5Y cells. SH-SY5Y cells were induced to overexpress the Ca²⁺-binding proteins calbindin and calmodulin at high titres using lentiviral particles. No changes in PSA-NCAM expression were observed in cells overexpressing Ca²⁺-binding proteins. Overall, mechanisms were identified by which PSA-NCAM regulation may become impaired in AD. This may provide novel therapeutic targets for enhancing impaired neuronal plasticity in AD which, in turn, may aid in the maintenance of cognition and delay disease onset.