Conference Agenda

Astrocytes represent one of the most abundant cell types in the central nervous system playing an essential role in almost every aspect of brain function. Although they have been for long considered a relatively homogeneous cell type, the idea that cortical astrocytes could constitute a various population at morphological, molecular and functional level, has been increasingly entertained in the last decade. Despite these recent advances, how this diversity originates and establishes during cortical development, remains largely unknown. Therefore, understanding the molecular mechanisms underlying the acquisition of specific astrocytic subtype identities represents a crucial step for gaining critical insights in how the brain develops and organizes in health and disease. Taking advantage of a cross-modal approach that combines in utero electroporation at different time points during cortical development, and high-throughput single-cell RNA sequencing, we identified five molecular distinct astrocyte subtypes. To understand how this molecular diversity originates, we performed trajectory inference analysis, revealing deferent temporal gene dynamics unique to each astrocyte subtype, which gives us insights into the maturation process of these cells during cortical development. Moreover, these astrocyte subtypes displayed a specific cortical localization that correlates to some extend with their time of birth: early- and late-born astrocytes tend to accumulate in the lower and upper parts of the cortical column, respectively. Altogether, these results unveil a novel logic underlying the diversity of this cell population in the cerebral neocortex.

Astrocytes’ heterogeneity is increasingly being recognized as crucial for a comprehensive understanding of the development and function of the CNS. While the main cerebellar astrocyte subtypes can be recognized based on morphology and location, little is known about their molecular identity, development, and functions. Single-cell RNA sequencing (scRNA-seq) represents a cutting-edge technique to address these questions. However, the available datasets have low astrocytes representation, hindering in-depth studies of these cells. We therefore integrated published scRNA-seq datasets with our original data of the developing and adult mouse cerebellum, enriched for astrocytes from distinct cerebellar regions. This resulted in comprehensive dataset of cerebellar astrocytes, covering several time points with an unprecedented spatial resolution. We uncovered the transcriptome of the main astrocyte types and highlighted that fate determination results from both cell intrinsic and extrinsic mechanisms. Moreover, differential expression, gene ontology, and pathway analyses revealed shared astrocyte-typical functions across all subtypes, driven by distinct gene sets, and divergent molecular and functional profiles across different cerebellar astrocyte subtypes. In this regard, the cellular localization within the cerebellar territory emerged as a critical factor in defining their transcriptional heterogeneity in the adult but not during early postnatal development. This, likely, reflects an unprecedented functional compartmentation within the astrocyte subtypes that may be driven by their specialized involvement in defined cerebellar circuits. Last, we could disclose several aspects of the ontogenesis and physiology of cerebellar nuclei astrocytes, so far essentially neglected. Indeed, our data pointed to their separate developmental trajectory compared to the other astrocyte subtypes and to a unique neurochemical profile, compatible with a modulatory role of cerebellar nuclei excitability. These findings represent a powerful source to investigate new aspects of cerebellar development, physiology and pathology and to understand astrocytes' role in these contexts, of high relevance for the development of novel therapeutic approaches.

Rett syndrome (RTT) is a rare devastating neurodevelopmental disorder that represents the most common genetic cause of severe intellectual disability in girls. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been reported in over 95% cases of classical forms of RTT. Initial studies supported a role for MeCP2 exclusively in neurons, which are profoundly defective in RTT. Indeed, RTT is considered a synaptopathy, characterized by dendritic spine dysgenesis, impaired spine plasticity, and disrupted excitatory/inhibitory balance. However, recent data supported the involvement also of astrocytes, which can affect neuronal maturation through non-cell autonomous mechanisms. Nevertheless, many aspects of RTT astrocyte dysfunctions remain still unknown and astrocytes’ heterogeneity in RTT has been only marginally explored. According to the crucial role of astrocytes in promoting synapse formation and functioning, we have investigated the influence of Mecp2 null astrocytes on synaptic phenotype. By using in vitro co-cultures, we have demonstrated that the lack of Mecp2 in cortical astrocytes dramatically affected the number of synaptic puncta in wild-type (WT) neurons. To gain insights into the involved molecular mechanisms, we performed bulk RNA-sequencing on WT neurons co-cultured with KO astrocytes; by profiling the molecular pathways activated in WT neurons by the paracrine effects triggered by KO cortical astrocytes, we confirmed that KO astrocytes influenced neuronal pathways mainly associated with synaptic maturation and functions, and elicited inflammatory responses. qPCR and luminex assay indicated the up-regulated release of a subset of cytokines by Mecp2 null astrocytes, with a possible synaptotoxic effect. We thus validated the role of one of these secreted factors, being aware that their identification might reveal novel therapeutic approaches for the treatment of RTT.

CETN2 is a centrosomal, calcium-binding protein implicated in mitosis and DNA repair best known for its expression in photoreceptors and ciliated cells. Here, we took advantage of a culture of human neuroepithelial stem (NES) to determine its expression and role in other cell types of the nervous tissue. We confirmed CETN2 centrosomal location in proliferating NES cells, and pointed out its relocation along with the differentiation process describing its selective cytoplasmic distribution in human astrocytes concomitantly with other known astroglia markers, while being absent in all other investigated cell types. These results were confirmed in human brain samples where we pointed out a peculiar CETN2-labeled astrocytes topography, not appreciable in murine tissues.

Next, we questioned about CETN2 role in pathologies in which aberrant calcium signaling and astrogliosis are part of the etiopathological process, finding in Alzheimer’s disease a prototypical condition. To this purpose, we examined CETN2 expression pattern in a cohort of control and sporadic AD patients investigating three brain areas differently hit by the disease: prefrontal and entorhinal cortices and the cerebellum.

By immunoblot, stereological analysis, and targeted-mass spectrometry we report a positive correlation between entorhinal CETN2 immunoreactivity and the neurocognitive impairment assessed by means of CDR and MMSE scores, as well as with the abundance of amyloid depositions and neurofibrillary tangles, thus highlighting a linear relationship between CETN2 expression and AD progression. We also investigated the co-expression of CETN2 with STAT3, NFATc3 and YKL-40, known reactive glia markers, and their abundance in proximity to plaques of severely affected entorhinal cortices.

Collectively our results provided not only the evidence that the novel astrocytic marker CETN2 is part of the astrocytic calcium toolkit undergoing rearrangements in AD, but also we identified CETN2 as an indicator of reactive astrogliosis.