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PDF file - 213K, Gene signatures distinguishing the different MB CSC populations segregate distinct human MB molecular subgroups, with MB1_EXCLUSIVE gene signature distinguishing the WNT molecular subgroup

PDF file - 183K, Long term-cultured hindbrain- and forebrain-derived NSCs are Sonic Hedgehog (Shh) pathway-independent, as cyclopamine treatment negatively affects the survival/proliferation of short-term cultured but not of long term-cultured NSCs

PDF file - 155K, Enforced expression of Ebf3 in CB WM and SVZ NSCs results in premature neuronal differentiation, under proliferative and differentiative conditions

PDF file - 157K, MB CSC lines are Sonic Hedgehog (Shh) pathway-independent, as their survival/proliferation is not affected by cyclopamine treatment and Smo silencing

PDF file - 104K, Details on experimental and bioinformatics procedures.

PDF file - 134K, Table S1 A. Lineage-specific differentiation of region-specific clonally derived NSCs is summarized as frequency of neurons, astrocytes and oligodendrocytes. B. Electrophysiological characteristics of neurons derived from the differentiation of region-specific NSCs. C. Analysis of the engraftment of region-specific clonally derived NSCs after transplantation in the early postnatal mouse cerebellum. Table S2 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in hindbrain (CB/IVv) or forebrain (SVZ) NSCs. Table S3 - Lineage-specific differentiation of clonally derived MB CSCs is summarized as frequency of neuron-, astrocyte- and oligodendrocyte-like cells. Table S4 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in tumorigenic Ptch+/- p53-/- MB CSCs vs. hindbrain NSCs (MB1_EXCLUSIVE), in non-tumorigenic Ptch+/- p53+/+ MB CSCs vs. hindbrain NSCs (MB2_EXCLUSIVE), and commonly expressed in both tumorigenic and non-tumorigenic CSCs vs. hindbrain NSCs (MB1_MB2 COMMON). Table S5 - Expression of EBF proteins in a collection of 33 human MB specimens as determined by immunohistochemistry.

PDF file - 130K, EBF3 expression can be retrieved in silico in human MBs by exploiting publicly available human data sets

PDF file - 132K, The gene signature containing the 297 genes differentially expressed between hindbrain- vs. forebrain-derived NSCs distinguishes the SHH molecular subgroup of human MBs

Despite the fact that several data have supported the involvement of the cerebellum in the functional alterations observed after prolonged cocaine use, this brain structure has been traditionally ignored and excluded from the circuitry affected by addictive drugs. In the present study, we investigated the effects of a chronic cocaine treatment on molecular and structural plasticity in the cerebellum, including BDNF, D3 dopamine receptors, ΔFosB, the Glu2 AMPA receptor subunit, structural modifications in Purkinje neurons and, finally, the evaluation of perineuronal nets (PNNs) in the projection neurons of the medial nucleus, the output of the cerebellar vermis. In the current experimental conditions in which repeated cocaine treatment was followed by a 1-week withdrawal period and a new cocaine challenge, our results showed that cocaine induced a large increase in cerebellar proBDNF levels and its expression in Purkinje neurons, with the mature BDNF expression remaining unchanged. Together with this, cocaine-treated mice exhibited a substantial enhancement of D3 receptor levels. Both ΔFosB and AMPA receptor Glu2 subunit expressions were enhanced in cocaine-treated animals. Significant pruning in Purkinje dendrite arborization and reduction in the size and density of Purkinje boutons contacting deep cerebellar projection neurons accompanied cocaine-dependent increase in proBDNF. Cocaine-associated effects point to the inhibitory Purkinje function impairment, as was evidenced by lower activity in these cells. Moreover, the probability of any remodelling in Purkinje synapses appears to be decreased due to an upregulation of extracellular matrix components in the PNNs surrounding the medial nuclear neurons.

All cerebellar GABAergic interneurons were derived from a common pool of precursor cells residing in the embryonic ventricular zone (VZ) and migrating in the prospective white matter (PWM) after birth, where both intrinsic and extrinsic factors contribute to regulate their amplification. Among the environmental factors, we focused on Sonic hedgehog (Shh), a morphogen well known to regulate neural progenitor cell proliferation. We asked if and how exogenous Shh treatment affects the lineage of cerebellar GABAergic interneurons. To address these issues, exogenous Shh was administered to embryonic and postnatal organotypic slices. We found that Shh is able to expand the pool of interneuron progenitors residing in the embryonic epithelium and in the postnatal PWM. In particular, Shh signalling pathway was highly mitogenic at early developmental stages of interneuron production, whereas its effect decreased after the first postnatal week. Gene expression analysis of sorted cells and in situ hybridization further showed that immature interneurons express both the Shh receptor patched and the Shh target gene Gli1. Thus, within the interneuron lineage, Shh might exert regulatory functions also in postmitotic cells. On the whole, our data enlighten the role of Shh during cerebellar maturation and further broaden our knowledge on the amplification mechanisms of the interneuron progenitor pool.

To remember our friend and colleague Ferdinando Rossi, prematurely passed away on 24th January 2014, a symposium was held during the ninth FENS meeting in Milan. It was focused on the development and plasticity of the cerebellum, the main topics of Ferdinando's research. From the talks of the invited speakers, Giacomo Consalez, Karl Schilling, Alain Chedotal, and Chris De Zeeuw, it clearly emerged that Ferdinando had a huge impact on the research of many scientists like them, as well as in the whole field of brain development and regeneration. With this symposium, we celebrated a brilliant scientist, devoted to Neuroscience with tireless passion and curiosity.

Stem cell lines that faithfully maintain the regional identity and developmental potency of progenitors in the human brain would create new opportunities in developmental neurobiology and provide a resource for generating specialized human neurons. However, to date, neural progenitor cultures derived from the human brain have either been short-lived or exhibit restricted, predominantly glial, differentiation capacity. Pluripotent stem cells are an alternative source, but to ascertain definitively the identity and fidelity of cell types generated solely in vitro is problematic. Here, we show that hindbrain neuroepithelial stem (hbNES) cells can be derived and massively expanded from early human embryos (week 5-7, Carnegie stage 15-17). These cell lines are propagated in adherent culture in the presence of EGF and FGF2 and retain progenitor characteristics, including SOX1 expression, formation of rosette-like structures, and high neurogenic capacity. They generate GABAergic, glutamatergic and, at lower frequency, serotonergic neurons. Importantly, hbNES cells stably maintain hindbrain specification and generate upper rhombic lip derivatives on exposure to bone morphogenetic protein (BMP). When grafted into neonatal rat brain, they show potential for integration into cerebellar development and produce cerebellar granule-like cells, albeit at low frequency. hbNES cells offer a new system to study human cerebellar specification and development and to model diseases of the hindbrain. They also provide a benchmark for the production of similar long-term neuroepithelial-like stem cells (lt-NES) from pluripotent cell lines. To our knowledge, hbNES cells are the first demonstration of highly expandable neuroepithelial stem cells derived from the human embryo without genetic immortalization.

Treatment options for degenerative cerebellar ataxias are currently very limited. A large fraction of such disorders is represented by hereditary cerebellar ataxias, whose familiar transmission facilitates an early diagnosis and may possibly allow to start preventive treatments before the onset of the neurodegeneration and appearance of first symptoms. In spite of the heterogeneous aetiology, histological alterations of ataxias often include the primary degeneration of the cerebellar cortex caused by Purkinje cells (PCs) loss. Thus, approaches aimed at replacing or preserving PCs could represent promising ways of disease management. In the present study, we compared the efficacy of two different preventive strategies, namely cell replacement and motor training. We used tambaleante (tbl) mice as a model for progressive ataxia caused by selective loss of PCs and evaluated the effectiveness of the preventive transplantation of healthy PCs into early postnatal tbl cerebella, in terms of PC replacement and functional preservation. On the other hand, we investigated the effects of motor training on PC survival, cerebellar circuitry and their behavioral correlates. Our results demonstrate that, despite a good survival rate and integration of grafted PCs, the adopted grafting protocol could not alleviate the ataxic symptoms in tbl mice. Conversely, preventive motor training increases PCs survival with a moderate positive impact on the motor phenotype.

By serving as the sole output of the cerebellar cortex, integrating a myriad of afferent stimuli, Purkinje cells (PCs) constitute the principal neuron in cerebellar circuits. Several neurodegenerative cerebellar ataxias feature a selective cell-autonomous loss of PCs, warranting the development of regenerative strategies. To date, very little is known as to the regulatory cascades controlling PC development. During central nervous system development, the proneural gene neurogenin 2 (Neurog2) contributes to many distinct neuronal types by specifying their fate and/or dictating development of their morphological features. By analyzing a mouse knock-in line expressing Cre recombinase under the control of Neurog2 cis-acting sequences we show that, in the cerebellar primordium, Neurog2 is expressed by cycling progenitors cell-autonomously fated to become PCs, even when transplanted heterochronically. During cerebellar development, Neurog2 is expressed in G1 phase by progenitors poised to exit the cell cycle. We demonstrate that, in the absence of Neurog2, both cell-cycle progression and neuronal output are significantly affected, leading to an overall reduction of the mature cerebellar volume. Although PC fate identity is correctly specified, the maturation of their dendritic arbor is severely affected in the absence of Neurog2, as null PCs develop stunted and poorly branched dendrites, a defect evident from the early stages of dendritogenesis. Thus, Neurog2 represents a key regulator of PC development and maturation.

In most CNS regions, the variety of inhibitory interneurons originates from separate pools of progenitors residing in discrete germinal domains, where they become committed to specific phenotypes and positions during their last mitosis. We show here that GABAergic interneurons of the rodent cerebellum are generated through a different mechanism. Progenitors for these interneurons delaminate from the ventricular neuroepithelium of the embryonic cerebellar primordium and continue to proliferate in the prospective white matter during late embryonic and postnatal development. Young postmitotic interneurons do not migrate immediately to their final destination, but remain in the prospective white matter for several days. The different interneuron categories are produced according to a continuous inside-out positional sequence, and cell identity and laminar placement in the cerebellar cortex are temporally related to birth date. However, terminal commitment does not occur while precursors are still proliferating, and postmitotic cells heterochronically transplanted to developing cerebella consistently adopt host-specific phenotypes and positions. However, solid grafts of prospective white matter implanted into the adult cerebellum, when interneuron genesis has ceased, produce interneuron types characteristic of the donor age. Therefore, specification of cerebellar GABAergic interneurons occurs through a hitherto unknown process, in which postmitotic neurons maintain broad developmental potentialities and their phenotypic choices are dictated by instructive cues provided by the microenvironment of the prospective white matter. Whereas in most CNS regions the repertoire of inhibitory interneurons is produced by recruiting precursors from different origins, in the cerebellum it is achieved by creating phenotypic diversity from a single source.

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.

Rationale Prior research has accumulated a substantial amount of evidence on the ability of cocaine to produce short- and long-lasting molecular and structural plasticity in the corticostriatal-limbic circuitry. However, traditionally, the cerebellum has not been included in the addiction circuitry, even though growing evidence supports its involvement in the behavioural changes observed after repeated drug experiences. Objectives In the present study, we explored the ability of seven cocaine administrations to alter plasticity in the cerebellar vermis. Methods After six cocaine injections, one injection every 48 h, mice remained undisturbed for 1 month in their home cages. Following this withdrawal period, they received a new cocaine injection of a lower dose. Locomotion, behavioural stereotypes and several molecular and structural cerebellar parameters were evaluated. Results Cerebellar proBDNF and mature BDNF levels were both enhanced by cocaine. The high BDNF expression was associated with dendritic sprouting and increased terminal size in Purkinje neurons. Additionally, we found a reduction in extracellular matrix components that might facilitate the subsequent remodelling of Purkinje-nuclear neuron synapses. Conclusions Although speculative, it is possible that these cocaine-dependent cerebellar changes were incubated during withdrawal and manifested by the last drug injection. Importantly, the present findings indicate that cocaine is able to promote plasticity modifications in the cerebellum of sensitised animals similar to those in the basal ganglia. This work was supported by grants and fellowships: Ministerio de Economía y Competitividad [PSI2011- 29181], FPI-PREDOC2009/05, FPU12/04059, PPF 2013 (13I087.01/1) and UJI (P1.1B2011-42).

© 2015 the authors. Cerebellar GABAergic interneurons in mouse comprise multiple subsets of morphologically and neurochemically distinct phenotypes located at strategic nodes of cerebellar local circuits. These cells are produced by common progenitors deriving from the ventricular epithelium during embryogenesis and from the prospective white matter (PWM) during postnatal development. However, it is not clear whether these progenitors are also shared by other cerebellar lineages and whether germinative sites different from the PWM originate inhibitory interneurons. Indeed, the postnatal cerebellum hosts another germinal site along the Purkinje cell layer (PCL), in which Bergmann glia are generated up to first the postnatal weeks, which was proposed to be neurogenic. Both PCL and PWM comprise precursors displaying traits of juvenile astroglia and neural stem cell markers. First, we examine the proliferative and fate potential of these niches, showing that different proliferative dynamics regulate progenitor amplification at these sites. In addition, PCL and PWM differ in the generated progeny. GABAergic interneurons are produced exclusively by PWM astroglial-like progenitors, whereas PCL precursors produce only astrocytes. Finally, through in vitro, ex vivo, and in vivo clonal analyses we provide evidence that the postnatal PWM hosts a bipotent progenitor that gives rise to both interneurons and white matter astrocytes.

Neurons in mammals do not undergo replicative aging, and, in absence of pathologic conditions, their lifespan is limited only by the maximum lifespan of the organism. Whether neuronal lifespan is determined by the strain-specific lifetime or can be extended beyond this limit is unknown. Here, we transplanted embryonic mouse cerebellar precursors into the developing brain of the longer-living Wistar rats. The donor cells integrated into the rat cerebellum developing into mature neurons while retaining mouse-specific morphometric traits. In their new environment, the grafted mouse neurons did not die at or before the maximum lifespan of their strain of origin but survived as long as 36 mo, doubling the average lifespan of the donor mice. Thus, the lifespan of neurons is not limited by the maximum lifespan of the donor organism, but continues when transplanted in a longer-living host.

The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2–/– interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.

Stem or progenitor cells acquire specific regional identities during early ontogenesis. Nonetheless, there is evidence that cells heterotopically transplanted to neurogenic regions of the developing or mature central nervous system may switch their fate to adopt host-specific phenotypes. Here, we isolated progenitor cells from different germinative sites along the neuraxis where GABAergic interneurons are produced (telencephalic subventricular zone, medial ganglionic eminence, ventral mesencephalon and dorsal spinal cord), and grafted them to the prospective white matter of the postnatal rat cerebellum, at the time when local interneurons are generated. The phenotype acquired by transplanted cells was assessed by different criteria, including expression of region-specific transcription factors, acquisition of morphological and neurochemical traits, and integration in the cerebellar cytoarchitecture. Regardless of their origin, all the different types of donor cells engrafted in the cerebellar parenchyma and developed mature neurons that shared some morphological and neurochemical features with local inhibitory interneurons, particularly in the deep nuclei. Nevertheless, transplanted cells failed to activate cerebellar-specific regulatory genes. In addition, their major structural features, the expression profiles of type-specific markers and the laminar placement in the recipient cortex did not match those of endogenous interneurons generated during the same developmental period. Therefore, although exogenous cells are influenced by the cerebellar milieu and show remarkable capabilities for adapting to the foreign environment, they essentially fail to switch their fate, integrate in the host neurogenic mechanisms and adopt clear-cut cerebellar identities.

The cerebellar circuits comprise a limited number of neuronal phenotypes embedded in a defined cytoarchitecture and generated according to specific spatio-temporal patterns. The local GABAergic network is composed of several interneuron phenotypes that play essential roles in information processing by modulating the activity of cerebellar cortical inputs and outputs. A major issue in the study of cerebellar development is to understand the mechanisms that underlie the generation of different interneuron classes and regulate their placement in the cerebellar architecture and integration in the cortico-nuclear network. Recent findings indicate that the variety of cerebellar interneurons derives from a single population of multipotent progenitors whose fate choices are determined by instructive environmental information. Such a strategy, which is unique for the cerebellum along the neuraxis, allows great flexibility in the control of the quality and quantity of GABAergic interneurons that are produced, thus facilitating the adaptive shaping of the cerebellar network to specific functional demands.

To elucidate the mechanisms that regulate neuronal placement and integration in the cerebellar circuitry, we assessed the fate of Purkinje cells transplanted to embryonic, juvenile and adult hosts, asking how architectural changes of the developing cortex influence their anatomical incorporation. Donor Purkinje cells navigate through the host parenchyma either along their natural migratory pathway or following unusual routes. In the latter case, donor neurons fail to orientate correctly and to establish the cortico-nuclear projection. Purkinje cells that follow the physiological route achieve the typical orientation and connectivity, but end displaced in the molecular layer if their arrival in the recipient cortex is delayed. Navigation routes and final settling of donor neurons vary with host age, depending on the ontogenetic construction of cortical layering, and particularly on the maturation of granule cells. The migratory behavior and homing of transplanted Purkinje cells is modified after external granular layer ablation, or neutralization of reelin signaling produced by granule cells. Therefore, although the cerebellar milieu remains receptive for Purkinje cells even after the end of development, correct placement of donor neurons depends on the timing of their migration, related to cerebellar developmental dynamics and granule cell layering.

Different cerebellar phenotypes are generated according to a precise spatiotemporal schedule, in which projection neurons precede local interneurons. Glutamatergic neurons develop from the rhombic lip, whereas GABAergic neurons originate from the ventricular neuroepithelium. Progenitors in these germinal layers are committed toward specific phenotypes already at early ontogenetic stages. GABAergic interneurons are thought to derive from a subset of ventricular zone cells, which migrate in the white matter and proliferate up to postnatal life. During this period, different interneuron categories are produced according to an inside-out sequence, from the deep nuclei to the molecular layer (we show here that nuclear interneurons are also born during late embryonic and early postnatal days, after glutamatergic and GABAergic projection neurons). To ask whether distinct interneuron phenotypes share common precursors or derive from multiple fate-restricted progenitors, we examined the behavior of embryonic and postnatal rat cerebellar cells heterotopically/heterochronically transplanted to syngenic hosts. In all conditions, donor cells achieved a high degree of integration in the cerebellar cortex and deep nuclei and acquired GABAergic interneuron phenotypes appropriate for the host age and engraftment site. Therefore, contrary to other cerebellar types, which derive from dedicated precursors, GABAergic interneurons are produced by a common pool of progenitors, which maintain their full developmental potentialities up to late ontogenetic stages and adopt mature identities in response to local instructive cues. In this way, the numbers and types of inhibitory interneurons can be set by spatiotemporally patterned signals to match the functional requirements of developing cerebellar circuits.

Cerebellar GABAergic projection neurons and interneurons originate from the ventricular neuroepithelium of the cerebellar primordium. However, while projection neurons are born within this germinal layer, interneurons derive from progenitors that delaminate into the prospective white matter. In spite of this common origin, the two main classes of GABAergic neurons are generated according to distinct strategies. Projection neurons are committed to their fate at early ontogenetic stages and acquire their mature phenotypes through cell-autonomous mechanisms. On the contrary, the different categories of cerebellar interneurons derive from a single pool of multipotent progenitors, whose fate choices, production rates and differentiation schedules are strongly influenced by environmental cues.