Conference Agenda

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-β1, a key mediator of survival at this transient stage. Constitutive Tns3 loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of Tns3 in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-β1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

In the developing mouse forebrain, temporally distinct waves of oligodendrocyte progenitor cells (OPCs) arise from different germinal zones and eventually populate either dorsal or ventral regions, where they present as transcriptionally and functionally equivalent cells. Despite that, here we show that the developmental origin of forebrain OPCs influences their responses to damage. Specifically, we found that DNA damage due to ablation of citron-kinase (Cit-K) or cisplatin treatment cell-autonomously disrupts OPC fate, resulting in cell death and senescence in the dorsal and ventral subsets, respectively. Such alternative fates are associated with the distinct developmental origins of forebrain dorsal vs. ventral OPCs, and with a different activation of NRF2-mediated anti-oxidant responses in the two cell subsets. In juvenile oligodendrocyte-specific Cit-K mutants, such OPC defects result in a severe and diffuse forebrain hypomyelination. Myelin defect is only party compensated in adult mice, where hypomyelination persists in the cerebral cortex – but not in other brain areas - and impacts on mouse cognitive ability.

Overall, these data indicate that, in presence of DNA damage, dorsal and ventral OPC subsets show a molecular diversity that makes them differentially vulnerable to pathological conditions and leads to region-specific and functionally-relevant long-term oligodendroglia dysfunctions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive motor neuron (MN) loss. Recent findings revealed an early role of oligodendrocyte (OL) dysfunction in disease progression. On this basis, restoring proper myelination and trophic support to MNs, by fostering oligodendrocyte precursor cell (OPC) maturation, may open new therapeutic perspectives for ALS. An important regulator of OPC differentiation is the P2Y-like GPR17 receptor. Our previous results revealed that an abnormal GPR17 expression was associated to OL dysfunction in the spinal cord of the SOD1G93A mouse model of ALS. Accordingly, primary spinal OPCs isolated from SOD1G93A mice displayed differentiation defects compared to wild-type cells, which were rescued by in vitro exposure to the non-selective GPR17 antagonist montelukast (MTK) [1]. Overall, these results suggest that the GPR17 receptor may represent a promising therapeutic target in ALS.

Here, we evaluated in vivo the effects of the oral administration of MTK at the dose of 10mg/kg/day and 30mg/kg/day, from symptom onset until end-stage, in SOD1G93A mice compared to vehicle-treated littermates. The results from the low-dose treatment did not match our expectations. On the other hand, the high-dose treatment significantly increased survival probability, delayed body weight loss, and ameliorated motor functions in female SOD1G93A mice, only. Noteworthy, in the spinal cord of female SOD1G93A mice, immunohistochemical analyses revealed that high-dose MTK administration significantly counteracted the pathological GPR17 upregulation, restored oligodendrocyte maturation, and induced significant changes in the reactive phenotype of microglia and astrocytes, suggesting pro-regenerative functions which may provide a permissive local environment thus preserving MNs from death and further supporting repair processes.

Globally, these results provide the first preclinical evidence indicating that repurposing of MTK may be a promising pharmacological strategy for ALS treatment.

Supported by AriSLA-Fondazione Italiana di ricerca per la SLA (2019)-grant-GPR17ALS-1 to MF and TB.

[1] PMID32244295.

Differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs) is a key event for axonal myelination in the brain. Remyelination occurs also in the adult brain, but during demyelinating pathologies, such as multiple sclerosis, this process is hindered. The neuromodulator adenosine is emerging as an important player in oligodendrogliogenesis, by activating all its metabotropic receptors (A₁R, A_2AR, A_2BR, A₃R). Previously, we contributed to demonstrating that selective stimulation of A_2BRs decreases OPC maturation in vitro, by inhibiting potassium currents necessary for their differentiation (Coppi et al., 2020).

The main purpose of this work was to explore the functional role of A_2BR on myelination processes in isolated dorsal root ganglion (DRG) neurons co-cultured with OPCs and in an animal model of demyelination, characterized by a cuprizone diet that induces OLs apoptosis in C57BL/6J mice.

The selective A_2BR agonist, BAY60-6583, increased myelin deposition on DRG-OPC co-culture, without affecting MBP expression level. Moreover, we found by immunocytochemical analysis that DRG neurons expressed A_2BR. These receptors are functionally implicated in DRG excitability since we demonstrated, by patch-clamp recordings, that the acute BAY60-6583-application increased the number of action potentials, an effect completely prevented in the presence of the selective A_2BR antagonist, PSB603.

After 5-weeks of the cuprizone diet, demyelination occurred in the corpus callosum (CC) and cerebral cortex (CTX), as indicated by the reduction in MBP expression levels. Chronic BAY60-6583-administration (i.p) for 2-weeks after the end of cuprizone, did not modify the demyelination index, indicating that their activation is not involved in this therapeutic time-window.

Data demonstrate that A_2BRs promote myelin deposition in OPC-DRG co-cultures, probably by increasing neuronal firing in DRG neurons, while are not involved in in vivo remyelination. These findings collected demonstrated an intricate role of the A_2BR in myelination processes, strictly dependent on their cellular localization.