Investigating coordination between stress-induced translational and transcriptional attenuation

Abstract

Neurodegeneration represents a significant health problem worldwide. Protein misfolding, mitochondrial dysfunction, and oxidative stress are all common features of neurodegeneration. To date, there is little therapeutic intervention available to tackle neurodegenerative disease. Protein misfolding results in the activation of several stress-induced processes including the integrated stress response (ISR) and stress-induced transcriptional downregulation (SITA). SITA and the ISR inhibit global transcription and translation respectively, while stress-induced transcripts escape both pathways and are upregulated. The ISR is physiologically relevant in neurodegenerative disease. Activation of the ISR is typified by phosphorylation of the α subunit on the eukaryotic initiation factor 2 (eIF2α), phospho-eIF2α (P-eIF2α) has been detected in a wide range of neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease and Huntington disease. SITA has also been evidenced in a Huntington’s disease model although little is known about the physiological relevance of SITA. Evidence of ISR activity in neurodegenerative disease models resulted in the production of a small molecule inhibitor of the ISR, ISR inhibitor (ISRIB). ISRIB treatment has been shown to improve age-related cognitive decline in mice. ISRIB treatment is also neuroprotective in prion diseased mice. Although ISRIB has not been shown as a human therapeutic, the use of ISRIB in mouse models exemplifies the importance of the ISR in neurodegenerative disease and brain activity. In this thesis, we consider the importance of both SITA and the ISR in neurodegenerative disorders and potential therapeutic intervention to inhibit both stress induced pathways in an effort to limit the disease-associated downregulation of gene expression. We also explore the relationship between ISR and SITA activation to elicit a coordinated pro-survival response. Results from this thesis show that SITA is inhibited by overexpression of the cyclin in the positive transcription elongation factor b (P-TEFb) complex, cyclin T1 (CCNT1), during acute heat shock (HS) and oxidative stress in cellulo. In identical conditions, CCNT1 overexpression has been shown to regulate the rate of ISR recovery. Multiple mechanisms of action have been suggested for further research; CCNT1 may interact with the ISR-induced activating transcription factor 4 (ATF4) and enhance transcription of genes that produce proteins involved in dephosphorylation of eIF2α or CCNT1 may directly interfere with ISR activation mechanisms. The translational impact of CCNT1 overexpression was also considered in basal, stress-free, conditions. Surprisingly, CCNT1 overexpression resulted in translational attenuation and ISR 3 activation in a cell cycle specific manner. A subset of G1 cells overexpressing CCNT1 show ISR activation. Although cell cycling has not been previously linked to the P-TEFb, the P-TEFb is required for the transcription of early G1 genes, facilitating the M/G1 transition in the cell cycle. Multiple hypotheses were suggested for further research based on this data, CCNT1 overexpression may increase cell proliferation and the rate of mitosis causing proteotoxic stress and ISR activation in daughter cells. Both overactivity of the P-TEFb and ISR activation are associated with therapy-resistant cancers, therefore these results may allude to understanding how the ISR becomes activated in cancers that present raised P-TEFb activity. Since CCNT1 overexpression is considered as a therapeutic intervention in this thesis, the impact of CCNT1 on the cell cycle and translation in the absence of stress must be considered when utilizing CCNT1 as a therapeutic. The mechanistic data in this study has been based on acute stress responses (1 hour of treatment with stressful stimuli). In neurodegenerative disease, the ISR is chronically activated. We therefore tested multiple neurodegenerative disease models for SITA activation. SITA was identified in a microglial cell line however could not be shown in primary murine brain mix culture, further research will identify stress response pathways in specific brain cell types to identify which cells in the brain react to stress by inhibiting transcription. Overall, this project shows the role of CCNT1 overexpression in SITA and the ISR during acute stress and shows the potential for manipulation of the P-TEFb to be used as a therapeutic in the context of neurodegenerative disease.