Periodic Reporting for period 1 - MOVEMeNt (Decoding alpha motor neurons diversity and selective vulnerability to disease)
Berichtszeitraum: 2020-02-01 bis 2022-01-31
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.
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.
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.