c-Myc dysregulation contributes to the glia-to-neuron miscommunication in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a non-cell-autonomous disease. In early 2000, seminal and concordant studies demonstrated that glia modulates disease progression in a mouse model expressing a mutant form of SOD1. This effect has not been validated yet in a transgenic mouse expressing TDP-43Q331K, so we selectively deleted the transgenic TDP-43 in astrocytes and observed an improvement of motor symptoms and a rescue of the cognitive impairment. We performed RNA sequencing of astrocytes purified from this mouse model, observing an increased expression of genes related to proliferation and de-differentiation at the early symptomatic stage. Additionally, we confirmed a relative increase in glial cell proliferation by Ki67 staining in vivo and by EdU staining in primary astrocytic cultures derived from this mouse model. By interrogating the genomes of 23.475 ALS patients versus 12.577 healthy controls, we revealed the enrichment of SNPs in the responsive elements of transcription factors linked to proliferation with a hub around c-myc. No differences were observed in c-Myc RNA and protein expression and stability in primary TDP-43Q331K astrocytes, while phosphorylated (active) c-Myc form was found to be increased by immunostaining. We observed this increase in several ALS models, including primary astrocytes expressing SOD1G93A, iNPC-derived astrocytes obtained from C9orf72 and sporadic ALS patients, and HEK293 cells overexpressing TDP-43. Interestingly, phosphorylated c-Myc form was found at higher levels than controls also in iPSCderived motoneurons expressing C9orf72, TDP-43M337V, SOD1I114T or derived from sporadic patients. c-Myc plays many functions in the cell, among which it enhances the release of extracellular vesicles (EVs), nanolipid structures shown to impact the non-cell autonomous degeneration in vitro. Our in vitro model showed a significant increase in the production of small EVs derived from ALS astrocytes. We observed that WT glia-derived EVs play a prosurvival effect on receiving neurons; this effect was lost when neurons were treated with gliaderived EVs from TDP-43Q331K cultures or WT cultures overexpressing c-Myc. Proteomic analysis revealed that ALS EVs and EVs derived from c-Myc-overexpressing cells were deficient in extracellular matrix proteins and integrins. Interestingly, the EVs isolated from TDP-43Q331K primary astrocytes were also less prone to enter recipient neurons. In addition, we notice that the pro-survival effect induced by WT glial EVs was lost on transgenic neurons, suggesting that receiving neurons carrying the mutation feature surface or endocytic alterations that prevent the transmission of the EV-induced prosurvival effect. To further characterize this hypothesis, we showed that HEK293 cells over-expressing TDP-43 feature impaired EV uptake and that iPSC-derived TDP-43M337V, SOD1I114T, and sporadic motoneurons feature altered endocytic pathways. All these data highlight alterations in the glial-to-neuron communication in ALS and suggest that the expression and function of integrins and extracellular matrix proteins should be further investigated in ALS, specifically focusing on rescuing neuronal endocytic pathways to achieve a functional treatment.