Unravelling the interplay between circadian clock and stress response using neurogenetics

About the Project

Molecular clock also interacts with a key kinase-signalling network called the integrated stress response (ISR) (1). Cellular stressors such as protein aggregates and reactive oxidative stress trigger ISR and modulate downstream gene transcription. Furthermore, altered ISR levels in fly neurons modify circadian rhythm, suggesting that the ISR is a cellular mechanism linking cellular stress to sleep-wake cycle. ISR can be triggered by misfolded protein aggregates that are associated with aged-related neurodegeneration. Initially ISR in the affected neurons eliminate the protein aggregates and the related cellular stresses, yet, prolonged cellular stress leads to neuronal dysfunction and cell death (1). Typically, the protein aggregates and the related ISR first affect specific brain areas. This selectivity is a widely observed phenomenon termed, selective vulnerability. However, the molecular mechanisms underlying selective vulnerability remain unclear.

The neuronal toxicity directed by aggregate-prone proteins including Ab, SCNA, HTT and TDP43 has been studied by expressing misfolded protein aggregate in Drosophila. Recently we found that the 150 so-called “clock neurons” that express the molecular clock in the fly brain are resistant to Ab-related ISR stress and toxicity (3). Intriguingly others found that clock neurons are more vulnerable to HTT-mediated toxicity (3).

Taken together, these findings suggest that the molecular clock regulates ISR stress to modify neuronal vulnerability to specific protein aggregates.

Aim: Using the versatile genetic and imaging tools available in the fruit fly, this PhD project aims to investigate the role of the ISR in shaping selectivity of the circadian clock against protein aggregates.

Objectives and Methods: the project will investigate the following question-oriented research objectives: 1. Which aggregates selectively cause ISR in clock neurons? Fly strains have been generated to express the aforementioned protein aggregates and the transgenic fluorescent reporters for ISR (4). The project will follow a protocol using techniques including brain dissection, immunostaining, confocal microscopy and image analysis. This protocol allows investigation of the fluorescent signals that indicates the extent of ER stress in the fly brains. Clock neurons will be immuno-labelled in the same brains in order to compare the levels of ER stress between clock and non-clock neurons. To further evaluate the direct effect of molecular clock on ISR, XBP1-GFP signals will be verified in aggregate expressing flies without molecular clock. 2. What candidate ISR related genes drives the selective vulnerability of clock neurons against protein aggregate? Using the publicly available RNASeq data and bioinformatics tools, the PhD student will look for candidate genes in the cellular stress related pathways. The identified genes and the key ISR genes will be knocked down in the fly clock neurons via somatic-CRISPR and RNAi technology and evaluate the effect of the knockdowns on ER stress level in the clock neurons and circadian behaviour. 3. Does arrhythmic ISR gene expression exacerbate cellular stress in neurodegeneration? Using smFISH/RNAscope technology, we will verify daily expression rhythm of ISR genes in the fly brains between wildtype and clock-gene mutant flies. The student will test if loss of the ISR gene rhythm modifies ISR levels flies expressing protein aggregates (reported by ATF4-dsRed).

Techniques that will be undertaken during the project:

Drosophila Genetics, Video-based behaviour assay, Ex vivo brain imaging,

Bioinformatics on RNA-Seq, Immunohistochemistry

BBSRC Strategic Research Priority: Understanding the Rules of Life – Neuroscience and behaviour

Start date: 25 September 2023

Entry requirements

• Those who have a 1st or a 2.1 undergraduate degree in a relevant field are eligible.
• Evidence of quantitative training is required. For example, AS or A level Maths, IB Standard or Higher Maths, or university level maths/statistics course.
• Those who have a 2.2 and an additional Masters degree in a relevant field may be eligible.
• Those who have a 2.2 and at least three years post-graduate experience in a relevant field may be eligible.
• Those with degrees abroad (perhaps as well as postgraduate experience) may be eligible if their qualifications are deemed equivalent to any of the above.
• University English language requirements apply

To apply

Carefully read the application advice on our website and submit your PhD application and MIBTP funding form.

https://le.ac.uk/study/research-degrees/funded-opportunities/bbsrc-mibtp