Studies of presenilin function in neurodegeneration and in human embryonic CNS during development

Abstract

Presenilin-1 (PS1) and -2 (PS2) gene mutations cause early onset familial Alzheimer's disease (FAD). Presenilins provide gamma-secretase activity for cleaving transmembrane proteins such as the amyloid precursor protein (APP), Notch receptors and ligands. Notch-1 activation by PS/gamma-secretase triggers expression of genes influencing neural cell fate during development and neurite outgrowth in differentiating neurons, suggesting that Notch signaling plays a role in maintaining stability of neurites and connections. Impaired PS and Notch function, involved in neural development, may contribute to AD neurodegeneration. This thesis explores the presence and function of AD-associated proteins in an animal model of neurodegeneration and during early human neurogenesis. The main emphasis was on PSs and Notch signalling components. Trimethyltin (TMT) intoxication induces slow and selective neuronal degeneration.

Paper I explored rat brain gene expression after TMT injection. In situ hybridisation was used to study levels of mRNAs for PS1, APP isoforms, and cfos and interleukin-10 (IL-1beta) at different times after TMT injection. Levels of APP 695 mRNA depicted TMT-induced neuronal loss, whereas expression of APP isoforms with the Kunitz protease inhibitor domain shifted from primarily neuronal expression to glial, PS1 mRNA expression did not change despite a loss of neurons. IL-1beta was localised to a few distinct glial cells. Apoptotic bodies were detected in the limbic system. Altered APP, APP-KPI, PS1, c-fos and IL-1beta expression after TMT administration suggests that this may be a useful model for studying features of AD-related gene expression.

Paper II studied the distribution of PS1 and Notch-1 receptor immunoreactivities (IRs) in human embryonic central nervous system (CNS) during the first trimester of development. The aim was to understand whether these proteins likely interact functionally during human foetal brain development. At very early stages of embryonic development intensive PS1-IR was seen predominantly in neurites in the ventral horn of the spinal cord, where it overlapped with neurofilament (NF)-200kDa-IR. PS1-IR was also seen in neuroepithelium of the ventricular zone of the tel- and mesencephalon, as well as brainstem. Notch-1-IR appeared in neuronal and ependymal cells throughout the CNS. Both proteins were localised in the neuroepithelial cell layer lining ventricles and in the cortical plate layer, where IRs were seen in the cell bodies. PS1-IR was also seen in thin neurites in the subplate of developing cortex. Thus although PSI and Notch-1 receptor are localised to the same differentiating cell populations in human cerebral cortex, making a direct interaction possible, these proteins are otherwise confined to different neurons or neuronal compartments, suggesting a role for PS1 during early CNS differentiation not involving Notch-1 processing.

Paper III compared the distribution of all four Notch receptor proteins and three ligands in the context of PS1 and PS2 co-localisation in the forebrain and spinal cord of human embryonic/foetal CNS. A divergent distribution of the different Notch receptor proteins was seen with only Notch-1 being co-localised with PS1 and PS2 in neuronal cells. Notch-2 and Notch-4 were not seen in the CNS at any age and in any region studied. Notch-3 expression was restricted to endothelial cells in blood vessels of both developing cortex and spinal cord. Jagged-1 was found sporadically in cells around the central canal within the spinal cord while Jagged-2 was not detected. Jagged-1 and Jagged-2 IRs were not found in the cortex. Jagged-1 was not co-distributed with either PS1 or PS2. Delta-1 ligand expression was seen in neuroepithelial cells of the ventricular zone of the cerebral cortex and in maturating neurons in the cortical plate and ventral horns of developing spinal cord. Notch-1, PS1 and Delta-1 in neuroepithelium of developing CNS suggests they may play a role during proliferation of progenitor neural cells. The strong IR of these three proteins in the cortical plate and in maturating neurons of spinal cord also suggests they regulate development of postmitotic neurons, dendrite growth and branching.

Paper IV investigated the role of PS1/gamma-secretase mediated Notch signaling in neuronal and glial differentiation of human embryonic neural stem and progenitor cells, grown in vitro as neurospheres. PS1 and Notch-1 expression correlated to the earliest appearance of neuronal or astrocyte markers, after induced differentiation. Cells began to migrate out of neurospheres after 2 h. Migrating cells were IR for both PS1 and Notch1 and the neural marker beta-tubulin III. Cells with features of astrocytes appeared after 8-24 h but only within the sphere. Neurospheres were also allowed to differentiate for 2, 8 or 24 h in the absence and presence of gammasecretase inhibitors. Differentiation into both neuronal and astrocyte phenotypes were promoted by y-secretase inhibition with DAPT or L-685,458. Both cell types appeared earlier and with more extensive outgrowth of cell processes after gamma-secretase inhibition. Developing neurons in treated neurospheres seemed also to be more mature in terms of size and length of neurites, and showed maintained levels of both PS1 and Notch-1 protein expression.

In conclusion, TMT intoxication lead to selective neurodegenerative changes and alterations in expression of genes involved in AD pathophysiology. The finding of the presence of PS1-IR in both the cell bodies and neurites of developing neurons strongly suggests divergent functions for PS1 during human brain development. These may include interactions with Notch-1, as well as Notch-independent mechanisms. PS1 and Notch-1 IR in differentiating neurons after PS1/gammasecretase inhibition also supports a role for PS1 in neurite growth involving y-secretase independent mechanisms. PS/gammasecretase-mediated Notch signaling may participate in maintaining neural stem cells in an undifferentiated state and in inhibition of neuronal differentiation and outgrowth. This could be relevant for improving cell replacement therapies in injury or disease.