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MacroAutophagy and Necrotic Neurodegeneration in Ageing

Periodic Reporting for period 2 - MANNA (MacroAutophagy and Necrotic Neurodegeneration in Ageing)

Reporting period: 2018-07-01 to 2019-12-31

Necrosis contributes critically in devastating human pathologies such as stroke, ischemia, and age-associated neurodegenerative disorders. Ageing increases susceptibility to neurodegeneration, in diverse species ranging from the lowly nematode Caenorhabditis elegans to humans. The mechanisms that govern necrotic neurodegeneration and its modulation by ageing are poorly understood. Autophagy has been implicated in necrosis and neurodegeneration, both with pro-survival and a pro-death roles. Autophagic flux declines with age, while induction of autophagy enhances longevity under conditions such as low insulin/IGF1 signalling and dietary restriction, which extend lifespan across diverse taxa. Our recent findings indicate that organelle-specific autophagy, including mitophagy, pexophagy and nucleophagy, is an important, evolutionarily conserved, determinant of longevity. This project aims to dissect the molecular underpinnings of neuron vulnerability to necrosis during ageing, focusing on cargo-specific macroautophagy. To this end, we are implementing a multifaceted approach that combines the power and versatility of C. elegans genetics with advanced, in vivo neuronal imaging and microfluidics technology. Our objectives are fourfold. First, monitor autophagic flux of organellar cargo, during neurodegeneration, under conditions that alter lifespan and identify mediators of organelle-specific autophagy in neurons. Second, conduct genome-wide screens for modifiers of age-inflicted neurodegeneration. Third, interrogate nematode models of human neurodegenerative disorders for organelle-specific autophagy and susceptibility to necrosis, upon manipulations that alter lifespan. Fourth, investigate the functional conservation of key mechanisms in mammalian models of neuronal necrosis. Together, these studies will deepen our understanding of age-related neurodegeneration and provide critical insights with broad relevance to human health and quality of life.
"We have completed work planned towards the first objective of the project: ""Monitor autophagic flux of organellar cargo, during neurodegeneration, under conditions that alter lifespan and identify mediators of organelle-specific autophagy in neurons"". Specifically we have:

1) Monitored autophagosome formation in neurons undergoing degeneration during ageing, in vivo and have assessed the requirement for specific autophagy genes in the process. We combined conventional fluorescence confocal microscopy techniques with cutting-edge, two-photon and second harmonic generation microscopy to follow and dissect intracellular alterations in dying C. elegans neurons during ageing, in vivo.
2) Developed and used both genetically encoded fluorescent markers, expressed exclusively in dying cells, and fluorescent dyes, to determine the fate of specific sub cellular organelles (mitochondria, lysosomes, the nucleus, the endoplasmic reticulum, the Golgi apparatus and peroxisomes), during necrosis.
3) Monitored intracellular physiological parameters such as pH, cytoplasmic calcium concentration and plasma membrane electrochemical potential in dying neurons.
4) Assessed animal metabolic parameters and phenotypes. These analyses were performed both in wild type animals and in mutants with altered lifespan. Extrinsic conditions that influence longevity, such as dietary restriction and chemical compounds were also examined.

We have initiated work towards the second objective: ""Genome-wide forward and reverse genetics screens for modifiers of age-inflicted necrotic neurodegeneration in C. elegans"". Specifically:

We are conducting both unbiased forward genetic screens for modifiers (suppressors or enhancers) of neurodegeneration, as well as genome-wide reverse genetic studies, based on RNAi technology. To facilitate screening, we developed and are currently using sensitised genetic backgrounds, in which the contribution of known components of the necrotic machinery is partially eliminated.

We have initiated work towards the third objective: ""Interrogation of nematode models of human neurodegenerative disorders for organelle-specific autophagy and susceptibility to necrosis, upon manipulations that alter lifespan"". Specifically:

We have established C. elegans models of human neurodegenerative disorders by overexpressing human genes implicated in disease, as well as, overexpress or mutate their nematode counterparts, in specific nematode neurons. Models that have been generated include excitotoxicity/stroke, Parkinson’s disease, Alzheimer’s disease, and pathologies associated with expanded polyglutamine repeat (polyQ) proteins. These models are currently used to examine susceptibility to neurodegenerative conditions during ageing. In addition, we are using tools and resources developed for Objective 1, to investigate the role of autophagy and organelle recycling in neurodegeneration.

We are in the process of setting up for the fourth objective: ""Assessment of the functional conservation of key cell death mechanisms in mammalian models of neuronal necrosis"". Specifically:

We are establishing a mammalian cell culture model of neuron necrosis, using hypoxic conditions, and treatment with chemicals, which will subsequently be used for pharmacological assays and RNAi experiments. We will then examine mammalian orthologues of nematode genes identified in forward and reverse genetic screens to be involved in necrosis for similar roles in mammals."
The project is at the forefront of research on the neurobiology of ageing, internationally. Cell death is a major biological process that plays pivotal roles in normal development, and homeostasis but, when either inappropriately implemented or blocked, contributes to severe pathological conditions. n humans, necrotic cell death generally occurs in response to severe changes of physiological conditions such as hypoxia and ischemia following stroke, hypoglycaemia, trauma and epilepsy. Neurodegenerative syndromes and diseases including Alzheimer, Huntington, Parkinson and amyotrophic lateral sclerosis also involve necrosis.

Successful completion of this project should yield the following novel and significant outcome:
1) Elucidation of physiological alterations associated with ageing that contribute to neuron necrosis.
2) Comprehensive identification of genes required for the execution of necrotic neuron death during ageing in C. elegans by genome-wide, reverse genetics approaches and unbiased forward genetic screens.
3) Genetic and molecular dissection of the role of organelle-specific autophagy in ageing-associated neurodegeneration.

In addition to planned studies, depending on the experimental outcome, we are poised to further exploit cutting-edge methodologies to manipulate neuronal function in vivo, during ageing in animals sensitized for neurodegeneration. Upon completion of the project, we should be able to describe in molecular detail how degeneration is initiated and what proteins are needed to enact death. Given that apoptotic cell death mechanisms are conserved between nematodes and humans, our analysis should lead to insights into the pathogenesis of human disease. Indeed, since a significant component of our proposed research is the establishment and dissection of simple-organism disease models, our project also contributes towards converting biological knowledge to practical applications, important to human health and quality of life.