ACSA-2 and GLAST classify subpopulations of multipotent and glial-restricted cerebellar precursors.

The formation of the cerebellum is highly coordinated to obtain its characteristic morphology and all cerebellar cell types. During mouse postnatal development, cerebellar progenitors with astroglial-like characteristics generate mainly astrocytes and oligodendrocytes. However, a subset of astroglial-like progenitors found in the prospective white matter (PWM) produces astroglia and interneurons. Characterizing these cerebellar astroglia-like progenitors and distinguishing their developmental fates is still elusive. Here, we reveal that astrocyte cell surface antigen-2 (ACSA-2), lately identified as ATPase, Na+/K+ transporting, beta 2 polypeptide, is expressed by glial precursors throughout postnatal cerebellar development. In contrast to common astrocyte markers, ACSA-2 appears on PWM cells but is absent on Bergmann glia (BG) precursors. In the adult cerebellum, ACSA-2 is broadly expressed extending to velate astrocytes in the granular layer, white matter astrocytes, and to a lesser extent to BG. Cell transplantation and transcriptomic analysis revealed that marker staining discriminates two postnatal progenitor pools. One subset is defined by the co-expression of ACSA-2 and GLAST and the expression of markers typical of parenchymal astrocytes. These are PWM precursors that are exclusively gliogenic. They produce predominantly white matter and granular layer astrocytes. Another subset is constituted by GLAST positive/ACSA-2 negative precursors that express neurogenic and BG-like progenitor genes. This population displays multipotency and gives rise to interneurons besides all glial types, including BG. In conclusion, this work reports about ACSA-2, a marker that in combination with GLAST enables for the discrimination and isolation of multipotent and glia-committed progenitors, which generate different types of cerebellar astrocytes.

Multiple origins and modularity in the spatiotemporal emergence of cerebellar astrocyte heterogeneity.

The morphological, molecular, and functional heterogeneity of astrocytes is under intense scrutiny, but how this diversity is ontogenetically achieved remains largely unknown. Here, by quantitative in vivo clonal analyses and proliferation studies, we demonstrate that the major cerebellar astrocyte types emerge according to an unprecedented and remarkably orderly developmental program comprising (i) a time-dependent decline in both clone size and progenitor multipotency, associated with clone allocation first to the hemispheres and then to the vermis(ii) distinctive clonal relationships among astrocyte types, revealing diverse lineage potentials of embryonic and postnatal progenitors; and (iii) stereotyped clone architectures and recurrent modularities that correlate to layer-specific dynamics of postnatal proliferation/differentiation. In silico simulations indicate that the sole presence of a unique multipotent progenitor at the source of the whole astrogliogenic program is unlikely and rather suggest the involvement of additional committed components.

Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia.

Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.

Sonic hedgehog patterning during cerebellar development.

The morphogenic factor sonic hedgehog (Shh) actively orchestrates many aspects of cerebellar development and maturation. During embryogenesis, Shh signaling is active in the ventricular germinal zone (VZ) and represents an essential signal for proliferation of VZ-derived progenitors. Later, Shh secreted by Purkinje cells sustains the amplification of postnatal neurogenic niches: the external granular layer and the prospective white matter, where excitatory granule cells and inhibitory interneurons are produced, respectively. Moreover, Shh signaling affects Bergmann glial differentiation and promotes cerebellar foliation during development. Here we review the most relevant functions of Shh during cerebellar ontogenesis, underlying its role in physiological and pathological conditions.

Oligodendrocyte Progenitors in Glial Scar: A Bet on Remyelination.

Oligodendrocyte progenitor cells (OPCs) represent a subtype of glia, giving rise to oligodendrocytes, the myelin-forming cells in the central nervous system (CNS). While OPCs are highly proliferative during development, they become relatively quiescent during adulthood, when their fate is strictly influenced by the extracellular context. In traumatic injuries and chronic neurodegenerative conditions, including those of autoimmune origin, oligodendrocytes undergo apoptosis, and demyelination starts. Adult OPCs become immediately activated; they migrate at the lesion site and proliferate to replenish the damaged area, but their efficiency is hampered by the presence of a glial scar-a barrier mainly formed by reactive astrocytes, microglia and the deposition of inhibitory extracellular matrix components. If, on the one hand, a glial scar limits the lesion spreading, it also blocks tissue regeneration. Therapeutic strategies aimed at reducing astrocyte or microglia activation and shifting them toward a neuroprotective phenotype have been proposed, whereas the role of OPCs has been largely overlooked. In this review, we have considered the glial scar from the perspective of OPCs, analysing their behaviour when lesions originate and exploring the potential therapies aimed at sustaining OPCs to efficiently differentiate and promote remyelination.

Oxysterols present in Alzheimer's disease brain induce synaptotoxicity by activating astrocytes: A major role for lipocalin-2.

Among Alzheimer's disease (AD) brain hallmarks, the presence of reactive astrocytes was demonstrated to correlate with neuronal loss and cognitive deficits. Evidence indeed supports the role of reactive astrocytes as mediators of changes in neurons, including synapses. However, the complexity and the outcomes of astrocyte reactivity are far from being completely elucidated. Another key role in AD pathogenesis is played by alterations in brain cholesterol metabolism. Oxysterols (cholesterol oxidation products) are crucial for brain cholesterol homeostasis, and we previously demonstrated that changes in the brain levels of various oxysterols correlate with AD progression. Moreover, oxysterols have been shown to contribute to various pathological mechanisms involved in AD pathogenesis. In order to deepen the role of oxysterols in AD, we investigated whether they could contribute to astrocyte reactivity, and consequently impact on neuronal health. Results showed that oxysterols present in mild or severe AD brains induce a clear morphological change in mouse primary astrocytes, accompanied by the upregulation of some reactive astrocyte markers, including lipocalin-2 (Lcn2). Moreover, astrocyte conditioned media analysis revealed a significant increase in the release of Lcn2, cytokines, and chemokines in response to oxysterols. A significant reduction of postsynaptic density protein 95 (PSD95) and a concurrent increase in cleaved caspase-3 protein levels have been demonstrated in neurons co-cultured with oxysterol-treated astrocytes, pointing out that mediators released by astrocytes have an impact on neurons. Among these mediators, Lcn2 has been demonstrated to play a major role on synapses, affecting neurite morphology and decreasing dendritic spine density. These data demonstrated that oxysterols present in the AD brain promote astrocyte reactivity, determining the release of several mediators that affect neuronal health and synapses. Lcn2 has been shown to exert a key role in mediating the synaptotoxic effect of oxysterol-treated astrocytes.

Cerebellar Astrocytes: Much More Than Passive Bystanders In Ataxia Pathophysiology.

Ataxia is a neurodegenerative syndrome, which can emerge as a major element of a disease or represent a symptom of more complex multisystemic disorders. It comprises several forms with a highly variegated etiology, mainly united by motor, balance, and speech impairments and, at the tissue level, by cerebellar atrophy and Purkinje cells degeneration. For this reason, the contribution of astrocytes to this disease has been largely overlooked in the past. Nevertheless, in the last few decades, growing evidences are pointing to cerebellar astrocytes as crucial players not only in the progression but also in the onset of distinct forms of ataxia. Although the current knowledge on this topic is very fragmentary and ataxia type-specific, the present review will attempt to provide a comprehensive view of astrocytes' involvement across the distinct forms of this pathology. Here, it will be highlighted how, through consecutive stage-specific mechanisms, astrocytes can lead to non-cell autonomous neurodegeneration and, consequently, to the behavioral impairments typical of this disease. In light of that, treating astrocytes to heal neurons will be discussed as a potential complementary therapeutic approach for ataxic patients, a crucial point provided the absence of conclusive treatments for this disease.

Automated segmentation of brain cells for clonal analyses in fluorescence microscopy images.

The understanding of how cell diversity within and across distinct brain regions is ontogenetically achieved is a pivotal topic in neuroscience. Clonal analyses based on multicolor cell labeling represent a powerful tool to tackle this issue and disclose lineage relationships, but produce enormous sets of fluorescence images, leading to time consuming analyses that may be biased by the operator's subjectivity. Thus, time-efficient automated software are needed to analyze images easily, accurately and without subjective bias. In this paper, we present a fully automated method, named FAST ('Fluorescent cell Analysis Segmentation Tool'), for the segmentation of neural cells labeled by multicolor combinations of fluorophores and for their classification into clones. The proposed method was tested on 77 high-magnification fluorescence images of adult mouse cerebellar tissues acquired using a confocal microscope. Automatic results were compared with manual annotations and two open-source software designed for cell detection in microscopic imaging. The algorithm showed very good performance in the cellular detection and in the assignment of the clonal identity. To the best of our knowledge, FAST is the first fully automated technique for the analysis of cellular clones based on combinatorial expression of fluorescent proteins. The proposed approach allows to perform clonal analyses easily, accurately and objectively, overcoming those biases and errors that may result from manual annotations. Moreover, it can be broadly applied to the quantification and colocalization within cells of fluorescent markers, therefore representing a versatile and powerful tool for automated quantitative analyses in fluorescence microscopy.