Astrocytes are multifunctional glial cells, implicated in neurogenesis and synaptogenesis, supporting and fine-tuning neuronal activity and maintaining brain homeostasis by controlling blood-brain barrier permeability. During the last years a number of studies have shown that astrocytes can also be converted into neurons if they force-express neurogenic transcription factors or miRNAs. Direct astrocytic reprogramming to induced-neurons (iNs) is a powerful approach for manipulating cell fate, as it takes advantage of the intrinsic neural stem cell (NSC) potential of brain resident reactive astrocytes. To this end, astrocytic cell fate conversion to iNs has been well-established in vitro and in vivo using combinations of transcription factors (TFs) or chemical cocktails. Challenging the expression of lineage-specific TFs is accompanied by changes in the expression of miRNAs, that post-transcriptionally modulate high numbers of neurogenesis-promoting factors and have therefore been introduced, supplementary or alternatively to TFs, to instruct direct neuronal reprogramming. The neurogenic miRNA miR-124 has been employed in direct reprogramming protocols supplementary to neurogenic TFs and other miRNAs to enhance direct neurogenic conversion by suppressing multiple non-neuronal targets. In our group we aimed to investigate whether miR-124 is sufficient to drive direct reprogramming of astrocytes to induced-neurons (iNs) on its own both in vitro and in vivo and elucidate its independent mechanism of reprogramming action. Our in vitro data indicate that miR-124 is a potent driver of the reprogramming switch of astrocytes towards an immature neuronal fate. Elucidation of the molecular pathways being triggered by miR-124 by RNA-seq analysis revealed that miR-124 is sufficient to instruct reprogramming of cortical astrocytes to immature induced-neurons (iNs) in vitro by down-regulating genes with important regulatory roles in astrocytic function. Among these, the RNA binding protein Zfp36l1, implicated in ARE-mediated mRNA decay, was found to be a direct target of miR-124, that be its turn targets neuronal-specific proteins participating in cortical development, which get de-repressed in miR-124-iNs. Furthermore, miR-124 is potent to guide direct neuronal reprogramming of reactive astrocytes to iNs of cortical identity following cortical trauma, a novel finding confirming its robust reprogramming action within the cortical microenvironment under neuroinflammatory conditions. In parallel to their reprogramming properties, astrocytes also participate in the maintenance of blood-brain barrier integrity, which ensures the physiological functioning of the central nervous system and gets affected contributing to the pathology of several neurodegenerative diseases. To study in real time the dynamic physical interactions of astrocytes with brain vasculature under homeostatic and pathological conditions, we performed 2-photon brain intravital imaging in a mouse model of systemic neuroinflammation, known to trigger astrogliosis and microgliosis and to evoke changes in astrocytic contact with brain vasculature. Our in vivo findings indicate that following neuroinflammation the endfeet of activated perivascular astrocytes lose their close proximity and physiological cross-talk with vasculature, however this event is at compensated by the cross-talk of astrocytes with activated microglia, safeguarding blood vessel coverage and maintenance of blood-brain integrity.

Dravet syndrome is a severe epileptic encephalopathy that begins during the first year of life and leads to severe cognitive and social interaction deficits. It is mostly caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes for the alpha-subunit of the voltage-gated sodium channel (Nav1.1) and is responsible mainly of GABAergic interneuron excitability. While different therapies based on the upregulation of the healthy allele of the gene are being developed, the dynamics of reversibility of the pathology are still unclear. In fact, whether and to which extent the pathology is reversible after symptom onset and if it is sufficient to ensure physiological levels of Scn1a during a specific critical period of time are open questions in the field and their answers are required for proper development of effective therapies. We generated a novel Scn1a conditional knock-in mouse model (Scn1aSTOP) in which the endogenous Scn1a gene is silenced by the insertion of a floxed STOP cassette in an intron of Scn1a gene; upon Cre recombinase expression, the STOP cassette is removed, and the mutant allele can be reconstituted as a functional Scn1a allele. In this model we can reactivate the expression of Scn1a exactly in the neuronal subtypes in which it is expressed and at its physiological level. Those aspects are crucial to obtain a final answer on the reversibility of DS after symptom onset. We exploited this model to demonstrate that global brain re-expression of the Scn1a gene when symptoms are already developed (P30) led to a complete rescue of both spontaneous and thermic inducible seizures and amelioration of behavioral abnormalities characteristic of this model. We also highlighted dramatic gene expression alterations associated with astrogliosis and inflammation that, accordingly, were rescued by Scn1a gene expression normalization at P30. Moreover, employing a conditional knock-out mouse model of DS we reported that ensuring physiological levels of Scn1a during the critical period of symptom appearance (until P30) is not sufficient to prevent the DS, conversely, mice start to die of SUDEP and develop spontaneous seizures. These results offer promising insights in the reversibility of DS and can help to accelerate therapeutic translation, providing important information on the timing for gene therapy delivery to Dravet patients.