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Decoding alpha motor neurons diversity and selective vulnerability to disease

Periodic Reporting for period 1 - MOVEMeNt (Decoding alpha motor neurons diversity and selective vulnerability to disease)

Reporting period: 2020-02-01 to 2022-01-31

Neurodegenerative diseases (ND), such as ALS, are highly debilitating diseases affecting the nervous system, more specifically the neurons responsible for movement, called motor neurons (MN). Although ND have different causes, onset, and prognosis, a common denominator includes selective vulnerability and consequential loss or dysfunction of neurons. Not all neurons are equally susceptible to disease as some neurons are more vulnerable and degenerate first, while others continue to function even at the last stages of the disease. The molecular reason beyond this selective vulnerability of specific MN subtype is not known. While many studies have greatly advanced our understanding on the molecular identities of neuronal subtypes, transcriptional signatures of the different subtypes are still critically missing. MOVEMeNt offers the first comprehensive analysis of the transcriptional profile of distinct MN classes on a genome-wide base. This is essential to uncover the molecular cues governing motor neuron vulnerability to ND. Unfortunately, there are limited techniques available to isolate distinct motor neuron types from the adult mouse, hampering the development of therapeutic treatment that specifically target vulnerable neurons.

The overall objective of the MOVEMeNt project is therefore to dissect the molecular fingerprints of vulnerable and resistant motor neuron subtypes to ultimately pinpoint at the determinants of vulnerability, instrumental for therapy development.

To this aim, MOVEMeNt project aimed to identify the molecular fingerprints of distinct MN subtypes in mouse adult spinal cords, otherwise impossible to be told apart without genetic labelling. I therefore developed a new methodology that leverage on the cell type-specific expression of markers to enrich the population of investigation for MN, while depleting it from other cell types, including astrocytes, microglia and oligodendrocytes. This enabled me to sample for rare cell types more frequently than we would be able to in an unbiased fashion.
I then characterized the isolated MN at the molecular level with a cellular resolution. Single nuclei sequencing analysis revealed the degree of diversity of MN in the spinal cord. I found several classes of neurons, of which molecular profile was subject of a deeper bioinformatic analysis. I have identified subtype-specific markers and gene assemblies that are likely important for the function of a specific subtype, ultimately revealing the subtype-specific molecular landscapes of distinct MN subtypes, including vulnerable and resistant MN. This will serve as a reference to determine the molecular substrate of selective disease vulnerability and instruct on novel candidates for the development of new successful therapeutic strategies.

Also, it is known that sprouting capacity is crucial for remodeling at the neuromuscular junctions, and to compensate for neuronal loss upon insult. Upon insult, vulnerable MN degenerate and do not retain sprouting capacity. In the MOVEMeNt project, I also aimed at deciphering the genome-wide transcriptomic profiles of “sproutogenic” and “non-sproutogenic” MN to extrapolate the molecular logics underpinning remodeling capacity. To this aim, vulnerable and resistant MN have been retrogradelly labelled by muscle-specific dye injection. This enabled us to visualize and isolate by laser capture microdissection distinct MN types having different sproutogenic capacity. Molecular analysis of the isolated MNs, will inform beyond the lack of remodeling capacity of vulnerable MN.

Taken together, MOVEMeNt provides the molecular substrate of vulnerability, which will serve as a pool of draggable candidates for new therapeutic strategies.
During these 24 months I have successfully achieved the identification and isolation of MN subtypes from the murine spinal cord, which was subsequently subjected to the analysis of their transcriptome at single cell resolution.
In particular, we have optimised a technique that enable to isolate specific populations of motor neurons, including those vulnerable to diseases, without employment of genetic reporter genes, rather based on the specific expression of subtype-specific markers. By engaging this technique we isolated and molecularly profiled nuclei (less fragile than the entire cell) of motor neurons from the murine spinal cord, which were subsequently subjected to single nuclei sequencing.
Bioinformatic analysis of the obtained transcriptomic profiles revealed the high degree of diversity of the neurons populating the spinal cord, pinpointing at specific molecular features of distinct motor neuron classes, which are known to have a peculiar susceptibility to insults, including neurodegenerative diseases. These unique fingerprints can inform on the molecular determinants of vulnerability, crucial for the identification of new candidates for therapy development.
We also undertook a different approach, were the entire has been analysed (rather than only nuclei. In particular, we have optimised a technique for laser microdissection of specific motor neurons that target different types of muscle. It is indeed known that upon insult, degenerating vulnerable neurons cannot reinnervate the muscle fibre, which remains denervated. In this scenario, the neighbouring resistant neurons, which retains sprouting capacity, extend an axon and try to compensate for neuronal loss by reinnervating the denervated fiber. To this aim, we injected fluorescent dies in specific muscles, which have then been retrogradely transported from the innervating motor neurons to the soma. Next, we were able to specifically dissect the single fluorescent neurons and distinguish vulnerable neurons (non-sprouting) from the resistant (sprouting) once. Comparison of sprouting and non-sprouting neurons is instrumental to the discovery of novel therapeutically draggable candidates. Results has been presented at national and international conferences, and several actions were taken to present the MOVEMeNt project to lay public, including middle and high schools.
Neurodegenerative diseases are devastating and affect millions of lives worldwide, imposing an increasing socio-economic burden. No cure is available. By identifying the molecular logics underpinning selective vulnerability of motor neurons, MOVEMeNt informs on potential candidates for therapy development. The obtained results are of high interest for many fields of investigation, e.g. basic neurobiology, study of MND, spinal cord injury, in vitro disease modeling, drug discovery. It is possible to investigate the class-specific expression of molecules known to contribute to the pathogenesis of neurodegenerative diseases, such as RNA binding proteins, molecules known to participate at the formation of stress granules, and/or dysregulation of genes known to be associated with MND in GWAS studies. This will ultimately pinpoint at novel therapeutically relevant pathways likely affected in late-onset diseases.
In addition, men seem to exhibit higher risk of contracting MND, earlier disease onset, and increased severity compared to women. In light of these considerations, MOVEMeNt will provide novel insight on the cell-autonomous differences between sex, uncovering the molecular basis of this phenomenon, and ultimately paving the way for personalized medicine. Finding a cue to ND would be a great benefit for the entire society.
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