The role of SQSTM1/P62 in axonal development and degeneration in an ALS-FTLD context

Young, Matthew J. (2020) The role of SQSTM1/P62 in axonal development and degeneration in an ALS-FTLD context. PhD thesis, University of Nottingham.

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Abstract

Amyotrophic lateral sclerosis and associated frontotemporal lobe dementia (ALS- FTLD) is a complex, fatal degenerative disease encompassing selective degeneration of upper and lower motor neurons, as well as neurons of the frontal and temporal brain regions, resulting in neuromuscular failure and dementia-like cognitive defects. In ALS-FTLD, disease progression is often rapid, with no effective cures currently available.

Recent studies have highlighted the contribution of dysfunctional autophagy to the development of neurodegenerative pathologies. In particular, the multi-functional autophagy receptor protein SQSTM1/P62 has been identified as a component of protein aggregates typically present in ALS-FTLD affected motor neurons. While P62 pathology has already been implicated in a wide variety of diseases, from cancer and metabolic diseases, to muscular-skeletal disorders, the P62L341V mutant has recently been identified as a hereditary ALS-FTLD mutation. Additionally, recent screens of FDA approved neuroleptics have highlighted the therapeutic potential of mTOR- independent autophagy activators like pimozide for ALS-related conditions. This study looks at the role that dysfunctional P62 mutations and autophagy regulation have in the axon – a highly specialised neuronal sub-unit, crucial to neuronal connectivity and thus function.

Pharmacological and transient transfection techniques were carried out in mouse primary cortical neurons. Cultures were subjected to autophagy modulating compounds, bafilomycin A1, rapamycin and pimozide, as well as P62 siRNA constructs, and EGFP-mCherry-P62 wild-type and loss-of-function mutant constructs. Images were obtained by Zeiss 200M microscope and analysed with FIJI and Prism 8.

In brief, this work has shown that the expression of the dysfunctional P62L341V mutant can produce differential outcomes, dependent on the stage of neuronal development. Mutant P62 expression in developing cortical neurons results in a phenotype of increased axonal outgrowth, replicated by pharmacological autophagy inhibition. In more mature cultures, this results in increased axonal degeneration. Regardless of axon maturity, our experiments show that mTOR-independent autophagy activation by pimozide can effectively rescue such phenotypes in these models. This contributes to the growing body of evidence linking autophagy dysfunction to ALS-FTLD pathology and neurodegenerative conditions, and deepens the understanding of the impacts of autophagy dysfunction on the axon.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dajas-Bailador, Federico
Layfield, Robert
Keywords: Autophagy, P62, neurobiology, axon biology
Subjects: Q Science > QP Physiology > QP351 Neurophysiology and neuropsychology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 61004
Depositing User: Young, Matthew
Date Deposited: 27 Aug 2020 14:05
Last Modified: 27 Aug 2020 14:15
URI: http://eprints.nottingham.ac.uk/id/eprint/61004

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