Translational control of mRNAs in the brain in health and disease: investigations into protein synthesis in the brain and related neuropsychiatric disorders

Abstract

Neuropsychiatric disorders, e.g. autism spectrum disorders and depression, present an increasing burden on society. Diagnoses are on the rise and despite a constantly increasing body of research, causes and mechanisms of disease generation remain elusive. To date, treatment is either difficult or unavailable. mRNA translation is an essential process for normal cell function. It is tightly regulated on both a global and local scale in cells. Local translation is particularly important for highly compartmentalised cells, such as neurons. mRNA translation is essential to the most basic processes in the brain, which include memory formation. Furthermore, dysregulation of translation, due to mutations in components of the translational machinery, has been shown to be both contributing and causal to some of the key phenotypes observed in neuropsychiatric disorders, e.g. autism spectrum disorders (ASD) or depression. For the work presented in my thesis, we employed a novel method based on deep sequencing, known as ribosome profiling, to quantitatively measure changes in mRNA ribosome occupancy, which can be used to predict changes in translation of individual transcripts at an omic scale. We applied ribosome profiling to a novel neuropsychiatric model resembling fragile X syndrome (FXS) phenotypes, TgMMP9. FXS is a genetic syndrome, in which patients show severe neurological and physiological symptoms and the currently most common known cause of ASDs. TgMMP9 is a mouse line overexpressing human matrix metalloproteinase 9 (MMP-9), conditionally in the brain. MMP-9 is a key molecule in the extracellular matrix of the brain and has been associated with memory, ASDs (FXS in particular), Alzheimer’s disease, and memory formation in the brain. We characterised translational regulation in TgMMP9 animals, using methods to study both global changes in translation and activation levels of known upstream regulators of translation. Furthermore, we carried out ribosome profiling of a well established mouse model of FXS, the Fmr1 knock-out. Likewise, we used ribosome profiling to study changes in translation in a novel mouse model of depression, eIF4ESer209Ala, entailing a mutation in an important molecular regulator of cap-dependent translation (eukaryotic initiation factor 4E, eIF4E), leaving the protein unphosphorylatable. Phosphorylation of eIF4E has previously been shown to be key in regulating transcript-specific translation. We also identified molecular pathways in these animals that impinge on translation and the dysregulation of which may in part be causative for the behavioural phenotypes we observe. Additionally, we identified genes important in early fear memory formation by carrying out ribosome profiling on hippocampal tissue from fear conditioned animals. To dissect the effect of the electrical shock and the actual memory formation, we profiled changes in mRNA expression and translation in two controls (naïve and shock only). We identified genes and confirmed their expression using quantitative real-time PCR, that change expression specifically in fearful memory formation. Finally, we adapted the ribosome profiling method for use in synaptoneurosomes, allowing us to study localised translation at synaptic terminals. In a brief experiment, we show that it is feasible to profile ribosome occupancy of mRNAs in biochemically isolated synaptic terminals, using two different protocols. This provides a powerful technique to study local translation at the synaptic compartment in both health and disease. Altogether, the work contained in this thesis, highlights the importance of mRNA translation regulation to the development of diverse neuropsychiatric disorders. We show regulation of specific subsets of mRNAs in these disorders both at a global and more local scale, as well as changes in the activation of pathways upstream of translation.