Role of activated transcription factor 4 (ATF4) in learning and memory

The aim of this study is to understand the role of Activated Transcription Factor 4 (ATF4) in the processes of learning and memory. The topic of learning and memory has always aroused great interest from time immemorial and although a lot of researches have been focused on this subject for a long time, many mechanisms have not yet been fully understood. Identifying the players and the mechanisms involved in learning and memory is of utmost importance because deficits in these cognitive functions are symptoms of common neurological diseases like stoke, depression, dementia and Alzheimer’s disease, one of the most wide spread neurodegenerative disease. It has already been established that new gene expression and protein synthesis are required for long term memory, providing the basis to think that transcription factors may play a key role in these processes. Several studies have demonstrated the involvement of different transcription factors in memory formation such as cAMP response element binding protein (CREB), CCAAT enhancer binding protein (C/EBP), activated protein 1 (AP1), early growth response factor (Egr) and Rel/nuclear factor kB (Rel/NFkB). Very little is known about the involvement of another transcription factor, Activated Transcription Factor 4. ATF4 is a member of the activated transcription factor (ATF)/cyclic AMP response element binding protein (CREB) family. It was originally described as a repressor of CRE-dependent gene transcription but recent studies have shown it to be a transcriptional activator. It is also a stress responsive gene, regulating the adaptation of cells to stress stimuli such as anoxia, endoplasmic reticulum stress and oxidative stress. ATF4 plays an essential role in development, and is particularly required for proper skeletal and eye development and is also involved in tumor progression and metastasis. ATF4 has always been reported as a memory repressor that blocks new gene expression required for memory formation but no study has ever investigated it in a specific and direct way. The aim of this thesis is to study, in a specific and direct manner, the role of ATF4 in the processes of learning and memory. To reach this goal, ATF4 expression was modified in mouse hippocampi, the brain region mainly involved in learning and memory, with the injection of lentivirus carrying ATF4 gene, for the gain-of-function analysis, and lentivirus carrying shATF4, for the loss-of-function studies. Before starting the experiments of ATF4 overexpression and downregulation, preliminary experiments were conducted to set up the injection coordinates to target the mouse hippocampi, to verify the lentiviral tropism and most importantly to evaluate the lentiviral spread, within the hippocampus, after the injection. The consequence of ATF4 gain- and loss-of-function was then studied in the performance of standard behavioral tests such as Water Maze tests and Fear Conditioning, widely used to assess spatial and associative memory respectively. The behavioral test results showed that ATF4 protein overexpression enhances spatial memory, under the weak training paradigm in the Morris Water Maze test, and associative memory while ATF4 downregulation impairs spatial memory under the standard training condition. After completing the behavioral tests, ATF4 overexpressed and downregulated mice were subjected to electrophysiological and neuronal spine analysis to verify if the alteration in cognitive functions, as a result of ATF4 modification, is supported by changes in synaptic potentiation and spine density and morphology. Long Term Potentiation (LTP) is a long lasting enhancement in neuronal transmission and is widely considered as a cellular model of learning and memory in the central nervous system. The long-term memory impairment of ATF4 downregulated mice is supported by electrophysiological analysis, in which ATF4 downregulated slices showed an impairment in LTP. Unexpectedly, LTP impairment was also found in ATF4 overexpressed slices, maybe due to the difference in the time between the injection and the behavioral tests or the electrophysiological recordings. Most of the intracellular pathways responsible for LTP require new gene expression and protein synthesis. This, in turn, leads to morphological changes required to sustain the enhancement of signal transmission. One of these morphological changes is the modification of the density and the morphology of dendritic spines. ATF4 up- and downregulation in hippocampal neurons does not affect spine density but ATF4 overexpression causes a significant increase in the percentage of mushroom spines as compared to that found after ATF4 downregulation. Mushroom spines with a large head are the most stable neuronal spines and contribute to strong synaptic connections, hence it has been hypothesized that they represent the “memory spines”. Collectively, these results support the hypothesis that the transcription factor ATF4 plays a positive role in synaptic plasticity and memory formation. Further studies need to be done to understand the molecular mechanisms through which ATF4 acts. This thesis represents only a step on the road towards understanding the complicate mechanisms of learning and memory, not forgetting that the most important discoveries were the result of small knowledge acquired step by step.