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Upregulated Autophagy – Possible answer to SCA3 Motor Neuron Disorder

Work done in the lab of Prof. Ravi Manjithaya at Autophagy Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research.

About author

Anushka Chakravorty

Anushka Chakravorty has obtained her Honours in B.Sc. Microbiology from St. Xavier’s College, Kolkata. Thereafter, she pursued her masters in Biotechnology from University of Pune, Maharashtra. She has joined as a Ph.D. research scholar at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR) in the laboratory of Dr. Ravi Manjithaya in close collaboration with Dr. Sheeba Vasu. Anushka is interested in understanding how autophagy is regulated in the presynaptic compartment of neurons with a major interest in unravelling the mechanisms that lead to synaptopathies.

Interview

How would you explain your research outcomes to the non-scientific community?

Neurons communicate with each other through synaptic connections and if these connections are lost or damaged, entire networks of neurons might get affected leading to brain damage. Damaged or dysfunctional synapses might subsequently lead to a class of neurodegenerative disorders, termed as synaptopathies. In our study, we showed that a fruit fly model of one such neurodegenerative condition, called Spinocerebellar Ataxia Type 3, is associated with motor neuron dysfunction and behavioural defects, such as locomotion deficits and reduced lifespan. In this particular neurodegenerative disorder (ND), a deubiquitinase called Ataxin-3 harbouring poly glutamine repeats, accumulates in the form of aggregates in or around the nucleus, leading to cellular dysfunction and pathology ranging from gait ataxia to peripheral muscular atrophy. To better understand such motor coordination defects, we studied the synapses formed by motor neurons (which are called neuromuscular junctions (NMJs)) in flies that harboured the mutant form of Ataxin-3 for detailed understanding. Indeed, we found impairment of NMJ morphology and function along with reduced proteostasis in these synapses. Autophagy is one of the important proteostasis pathways in the synapses. In this pathway, double-mem

Figure 1: Third instar larvae expressing truncated form of Ataxin-3 protein containing either 27 (non-pathogenic) or 78 (pathogenic) polyQ repeats under the motor neuron specific driver D42-Gal4 were subjected to behavioural assays and simultaneous assessment of phenotypic and functional defects in NMJs.
Figure 1: Third instar larvae expressing truncated form of Ataxin-3 protein containing either 27 (non-pathogenic) or 78 (pathogenic) polyQ repeats under the motor neuron specific driver D42-Gal4 were subjected to behavioural assays and simultaneous assessment of phenotypic and functional defects in NMJs.

braned vesicles called autophagosomes form de novo around the misfolded protein cargo and capture it. These vesicles then fuse with lysosomes to degrade the cargo, and the contents are released into the cytoplasm for reuse. Interestingly, we overexpressed one of the key core autophagy proteins, called Atg8a tissue-specifically in the motor neurons, and found that the behavioural deficits and synaptic dysfunction were partially rescued. Thus, our genetically amenable system provides a tool to enhance our understanding of synaptopathies for not just polyglutamine repeat-associated neurodegenerative diseases, but motor-neuron disorders in general.

Figure 2: Ubiquitous expression of non-pathogenic Ataxin-3 in cell bodies of motor neurons in the ventral nerve cord of third instar Drosophila larvae (top panel) versus aggregation of pathogenic Ataxin-3 into distinct foci in the cell bodies of the ventral nerve cord (bottom panel). Note how pathogenic Ataxin-3 is absent from the long axons emanating from the nerve cord.
Figure 2: Ubiquitous expression of non-pathogenic Ataxin-3 in cell bodies of motor neurons in the ventral nerve cord of third instar Drosophila larvae (top panel) versus aggregation of pathogenic Ataxin-3 into distinct foci in the cell bodies of the ventral nerve cord (bottom panel). Note how pathogenic Ataxin-3 is absent from the long axons emanating from the nerve cord.

How do these findings contribute to your research area?

Peripheral nervous system dysfunction is not well understood for many neurodegenerative conditions that are associated with gait ataxia and parkinsonism. Through our study, we have characterized a model for studying synapse dysfunction of motor neurons, which can be further utilized for carrying out genetic and/or pharmacological screens to identify potential therapeutic targets against synaptopathies.

“We have characterized a model for studying synapse dysfunction of motor neurons, which can be further utilized for carrying out genetic and/or pharmacological screens to identify potential therapeutic targets against synaptopathies. ”

What was the exciting moment during your research?

It is indeed difficult to pinpoint to ‘the exciting moment’ during my journey because there were several. Observing the beauty of the microscopic world in the synapses of a fruit fly still makes my day. I particularly remember the incident when I was able to first observe these beautiful NMJs spanning the entirety of the musculature of fly larvae. It was an exhilarating moment for me. When I started this project, every tiny observation (such as video graphing the fly larvae to observe defects associated with their locomotion) was sensational in its own ways. Another thrilling moment was being able to observe the autophagosomes being trafficked along the axons of motor neurons using live cell microscopy in immobilized larvae.

What do you hope to do next?

I hope to carry forward these findings to address research problems that are endemic to India. We are now on the way to understanding how synaptic autophagy pathway is regulated in synapses through a genetic screen in Drosophila, these targets would be later used for uncovering molecular mechanisms underlying ataxias’ endemic in our country.

Where do you seek scientific inspiration from?

There are several people who have inspired me throughout my Ph.D. journey including my mentor- Dr. Ravi Manjithaya, my co-PI- Dr. Sheeba Vasu, my colleagues, parents and many more people who have directly or indirectly kindled the flame of curiosity to explore the vast unknowns with respect to understanding the nuances of synaptic biology. Reading about the work of scientists who unravelled the mysteries and complexities of the tiny cell years ago when technology was not as advanced as in present days, is also something that motivates me.

How do you intend to help Indian science improve?

I sincerely thank the Indian government and all funding agencies for their financial support to carry out my research project. However, I do think that for Indian science to improve and reach its pinnacle, the focus should be on fund allocations in both translational and basic research along with encouraging strong collaborations with researchers from inter-disciplinary fields.

Personally, given an opportunity, I would like to contribute to communication and outreach programs for school level students to educate them about the opportunities in this field and inculcate the curiosity required for scientific advancement in general.

Reference

Chakravorty, A., Sharma, A., Sheeba, V., and Manjithaya, R. (2022). Glutamatergic Synapse Dysfunction in Drosophila Neuromuscular Junctions Can Be Rescued by Proteostasis Modulation. Front Mol Neurosci 15, 842772. doi: 10.3389/fnmol.2022.842772. https://www.frontiersin.org/articles/10.3389/fnmol.2022.842772/full

Copy Editor: Pratibha Siwach

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