In this rotation you will learn techniques for the purification of extracellular vesicles (EV and exosomes) from cultured mammalian cells. Protein content will be catalogued by state-of-the-art mass spectrometry analyses. Candidate proteins identified will be quantified by western blotting and bioinformatics analyses will be used to uncover possible sequence ‘signatures’ of EV components.
School of Life Sciences
Main Supervisor: Sebastien Serres
Oxidative stress and dysfunction of autophagy are the main features of motor neuron (MN) disorders, including amyotrophic lateral sclerosis (ALS). In this pathology, the progressive loss of MNs in the brain and spinal cord leads to muscle wasting and respiratory failure, which ultimately causes death. Oxidative stress occurs in familial form of ALS and is associated with mutations in SOD1, a gene that encodes the anti-oxidant enzyme superoxide dismutase. The major pathological feature of sporadic ALS is dysfunction of TDP-43 protein degradation. Recent data suggests that a common mechanism in ALS toxicity may be in play for both forms, involving non-neuronal cells (for example, astrocytes). In normal conditions, astrocytes mediate neuro-inflammatory responses in the brain and spinal cord to protect MNs against disease or injury. However, when stressed by oxidants or protein inclusions as seen in ALS, astrocytes become reactive and promote MN toxicity. Neuro-inflammatory cytokines normally stimulate signal transducer and activator of transcription 3 (STAT3), which is instrumental for astrocyte reactivity by preserving energy supply, redox balance, and cell viability. Interestingly, STAT3 expression can be modulated by oxidants (for example, reactive oxygen species (ROS)) or extracellular vesicles (EVs) containing ubiquitinated protein (for example, TDP-43). Based on this evidence, we hypothesise that in both familial and sporadic ALS, elevated levels of oxidised STAT3 or accumulation of EVs in astrocytes may significantly modulate the spectrum of expressed STAT3 target genes. This, in turn, could cause an imbalance between STAT3 signalling modes in astrocytes and therefore contribute to toxicity and MN loss.
In this PhD project, we will identify the mechanism by which neuronally-derived EVs modulate STAT3 responses to cytokine signalling in astrocytes and how this impacts on astrocyte reactivity (for example, metabolic changes). Focus will be on the role of neuronal autophagy failure and its relationship to the enhanced formation of TDP-43-containing EV. To this end, we will examine STAT3 post-translational modifications, DNA interactions, and gene expression profiles in primary murine astrocytes (PMAs) and their impact on metabolic changes in PMAs using state-of-the art nuclear magnetic resonance (NMR) spectroscopy and 13C stable isotopes. We will correlate these changes with normal STAT3 responses to cytokine signalling and determine how this affects MN survival. We will also identify gene and proteins profiles that are associated with oxidative stress and EVs fractions, respectively. This data will be used to extract the genetic and proteomic drivers for the phenotypic change in astrocytes, which can then be compared with other available data from human/murine astrocytes.
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