Ral GTPases are critical regulators of spinal cord myelination and homeostasis.

Efficient myelination supports nerve conduction and axonal health throughout life. In the central nervous system, oligodendrocytes (OLs) carry out this demanding anabolic duty in part through biosynthetic pathways controlled by mTOR. We identify Ral GTPases as critical regulators of mouse spinal cord myelination and myelin maintenance. Ablation of Ral GTPases (RalA, RalB) in OL-lineage cells impairs timely onset and radial growth of developmental myelination, accompanied by increased endosomal/lysosomal abundance. Further examinations, including transcriptomic analyses of Ral-deficient OLs, were consistent with mTORC1-related deficits. However, deletion of the mTOR signaling-repressor Pten in Ral-deficient OL-lineage cells is unable to rescue mTORC1 activation or developmental myelination deficiencies. Induced deletion of Ral GTPases in OLs of adult mice results in late-onset myelination defects and tissue degeneration. Together, our data indicate critical roles for Ral GTPases to promote developmental spinal cord myelination, to ensure accurate mTORC1 signaling, and to protect the healthy state of myelin-axon units over time.

SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia.

Friedreich's ataxia (FRDA) is an untreatable disorder with neuro- and cardio-degenerative progression. This monogenic disease is caused by the hyper-expansion of naturally occurring GAA repeats in the first intron of the FXN gene, encoding for frataxin, a protein implicated in the biogenesis of iron-sulfur clusters. As the genetic defect interferes with FXN transcription, FRDA patients express a normal frataxin protein but at insufficient levels. Thus, current therapeutic strategies are mostly aimed to restore physiological FXN expression. We have previously described SINEUPs, natural and synthetic antisense long non-coding RNAs, which promote translation of partially overlapping mRNAs through the activity of an embedded SINEB2 domain. Here, by in vitro screening, we have identified a number of SINEUPs targeting human FXN mRNA and capable to up-regulate frataxin protein to physiological amounts acting at the post-transcriptional level. Furthermore, FXN-specific SINEUPs promote the recovery of disease-associated mitochondrial aconitase defects in FRDA-derived cells. In summary, we provide evidence that SINEUPs may be the first gene-specific therapeutic approach to activate FXN translation in FRDA and, more broadly, a novel scalable platform to develop new RNA-based therapies for haploinsufficient diseases.

RNA activation of haploinsufficient Foxg1 gene in murine neocortex.

More than one hundred distinct gene hemizygosities are specifically linked to epilepsy, mental retardation, autism, schizophrenia and neuro-degeneration. Radical repair of these gene deficits via genome engineering is hardly feasible. The same applies to therapeutic stimulation of the spared allele by artificial transactivators. Small activating RNAs (saRNAs) offer an alternative, appealing approach. As a proof-of-principle, here we tested this approach on the Rett syndrome-linked, haploinsufficient, Foxg1 brain patterning gene. We selected a set of artificial small activating RNAs (saRNAs) upregulating it in neocortical precursors and their derivatives. Expression of these effectors achieved a robust biological outcome. saRNA-driven activation (RNAa) was limited to neural cells which normally express Foxg1 and did not hide endogenous gene tuning. saRNAs recognized target chromatin through a ncRNA stemming from it. Gene upregulation required Ago1 and was associated to RNApolII enrichment throughout the Foxg1 locus. Finally, saRNA delivery to murine neonatal brain replicated Foxg1-RNAa in vivo.

Upregulating endogenous genes by an RNA-programmable artificial transactivator.

To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action.

Reprogramming fibroblasts to neural-precursor-like cells by structured overexpression of pallial patterning genes.

In this study, we assayed the capability of four genes implicated in embryonic specification of the cortico-cerebral field, Foxg1, Pax6, Emx2 and Lhx2, to reprogramme mouse embryonic fibroblasts towards neural identities. Lentivirus-mediated, TetON-dependent overexpression of Pax6 and Foxg1 transgenes specifically activated the neural stem cell (NSC) reporter Sox1-EGFP in a substantial fraction of engineered cells. The efficiency of this process was enhanced up to ten times by simultaneous inactivation of Trp53 and co-administration of a specific drug mix inhibiting HDACs, H3K27-HMTase and H3K4m2-demethylase. Remarkably, a fraction of the reprogrammed population expressed other NSC markers and retained its new identity, even after switching off the reprogramming transgenes. When transferred into a pro-differentiative environment, Pax6/Foxg1-overexpressing cells activated the neuronal marker Tau-EGFP. Frequency of Tau-EGFP positive cells was almost doubled upon delayed delivery of Emx2 and Lhx2 transgenes. A further improvement of the neuron-like cell output was achieved by inhibition of the BMP and TGFß pathways. Tau-EGFP positive cells were able to generate action potentials upon injection of depolarizing current pulses, further indicating their neuron-like phenotype.