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Exploiting single-cell RNA sequencing to clarify the interplay of neurons and glia in neurodegeneration

Primary Supervisor: Professor Flaviano Giorgini, Department of Genetics and Genome Biology

Secondary supervisor: Dr Roberto Feuda

PhD project title: Exploiting single-cell RNA sequencing to clarify the interplay of neurons and glia in neurodegeneration

University of Registration: University of Leicester

Project outline:

The loss or dysfunction of specific cell populations underlies the pathogenesis of many debilitating brain diseases known collectively as neurodegenerative disorders. While to date the predominant focus in these disorders has been perturbations within neuronal populations, glial cells such as astrocytes, oligodendrocytes and microglia are likely to contribute to disease onset and progression. a-synucleinopathies – which include Parkinson’s disease (PD) - are a subset of neurodegenerative disorders which feature pathological accumulation of the protein a-synuclein into proteinaceous deposits in cells of the central nervous system. Notably, the gene encoding a-synuclein – SNCA – is linked to autosomal dominantly inherited forms of PD, with certain genetic variants also identified as risk factors. PD is the second most common neurodegenerative disorder, with patients exhibiting rigidity, bradykinesia (slowness of movement) and tremors. These symptoms are likely due to dysfunction or loss of dopamine-producing neurons with the substantia nigra pars compacta region of the midbrain, as evidenced in port-mortem analysis of brains from individuals with PD. In the context of PD and the related disorder dementia with Lewy bodies, misfolded a-synuclein accumulates in intraneuronal structures known as Lewy bodies. Notably, internalisation of a-synuclein by astrocytes and microglia is thought to contribute to the spread of pathology. Relatedly, in the nervous system disorder multiple systems atrophy, oligodendrocytes exhibit cytoplasmic a-synuclein aggregates.

The focus of this project will be dissecting the neuronal and glial contributions to a-synucleinopathies, using the fruit fly Drosophila melanogaster as a model. This work will take advantage of the powerful bitransgenic GAL4-UAS system to target expression of a-synuclein in discrete neuronal and glial cell populations within the fruit fly to ascertain the cell intrinsic and extrinsic gene expression alterations. Specifically, the objectives of this PhD studentship and the methods to be employed are:

  • To analyze recently generated single-cell datasets to determine neuronal subtype and glial specific genetic drivers.
  • Use the cell-type specific genetic drivers to drive the expression of a-synuclein in specific populations of neurons and glia in the brain of Drosophila melanogaster using transgenic approaches combined with classical genetics
  • Perform fluorescence-activated cell sorting (FACS) flow cytometry to separate specific cell types with the brain and isolate total RNA from these populations
  • Undertake single-cell RNA sequencing and expression analyses to determine cell-autonomous and non cell-autonomous alterations in gene expression in the selected cell types
  • Determine from these analyses candidate pathways/genes to dissect the potential cell-type specific roles of a-synuclein in pathogenesis

References:

  1. Mechanisms of Parkinson's Disease: Lessons from Drosophila. Hewitt VL, Whitworth AJ. Curr Top Dev Biol. 2017;121:173-200. doi: 10.1016/bs.ctdb.2016.07.005.
  2. Neurons and Glia Interplay in alpha-Synucleinopathies. Mavroeidi P, Xilouri M. Int J Mol Sci. 2021 May 8;22(9):4994. doi: 10.3390/ijms22094994.
  3. Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour. Breda C, Nugent ML, Estranero JG, Kyriacou CP, Outeiro TF, Steinert JR, Giorgini F. Hum Mol Genet. 2015 Feb 15;24(4):1077-91. doi: 10.1093/hmg/ddu521.
  4. Single cell transcriptomics comes of age. Aldridge S, Teichmann SA. Nat Commun. 2020 Aug 27;11(1):4307. doi: 10.1038/s41467-020-18158-5.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour & Integrated Understanding of Health: Ageing

Techniques that will be undertaken during the project:

  • Drosophila genetics and husbandry
  • Single-cell RNA sequencing
  • FACS analysis (cell sorting)
  • Bioinformatics analyses
  • Immunohistochemistry
  • Advanced microscopy

Contact: Professor Flaviano Giorgini, University of Leicester