dc.description.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. | en |
dc.relation.hasversion | Inês Silva Amorim, Sonal Kedia, Stella Kouloulia, Konstanze Simbriger, Ilse Gantois, Seyed Mehdi Jafarnejad, Yupeng Li, Agniete Kampaite, Tine Pooters, Nicola Romanò, and Christos G. Gkogkas Loss of eIF4E phosphorylation engenders depression-like behaviors via selective mRNA translation, Journal of Neuroscience, 24 January 2018, 2673-17; DOI: 10.1523/JNEUROSCI. 2673-17.2018, | en |