Search

Supplementary Figure from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Supplementary Table from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

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

Supplementary Figure Legends

Supplementary Figure 1: Etv1 is required for intramuscular and myenteric ICC development. Supplementary Figure 2: Etv1 ablation with tamoxifen decreases Etv1 and Kit expression in Kit^delta558/+;Etv1^flox/flox;Rosa26^CreERT2/CreERT2 mice. Supplementary Figure 3: Etv1 positively regulates Kit expression in murine stomach. Supplementary Figure 4: ETV1 regulates KIT expression in GIST48 cells. Supplementary Figure 5: MAP kinase pathway inhibition decreases ETV1 target gene expression. Supplementary Figure 6: Dual lineage inhibition inhibits tumor initiation in vivo.

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

Supplementary Table 2: Gene sets enriched among genes DOWNregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

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

Supplementary Table 3: Gene sets enriched among genes UPregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

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.

Supplementary Table 1: Differentially expressed genes (p1.5) between oil and tamoxifen treated tumors to conditionally ablate Etv1.

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

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a Kit

Introduction: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit receptor. While highly effective, tyrosine kinase inhibitors like imatinib are not curative. The natural ligand for the Kit receptor is Kit Ligand (KitL), which exists in both soluble and membrane-bound forms. We sought to determine the role of KitL in the oncogenic Kit signaling of GIST. Methods: KitV558Δ/+ mice, which spontaneously develop an intestinal GIST, were treated with imatinib or vehicle for 1 week, and sorted non-immune (CD45-) cells from tumors were submitted for single-cell RNA sequencing (n=3/group). GIST T1 cells were treated with KitL with or without imatinib and oncogenic Kit signaling was evaluated. KitV558Δ/+ mice were crossed with various KitL mutant and inducible models and tumor weights were measured (n=3-4/group). Human GISTs were stained for KitL expression by IHC, and an existing bulk RNA sequencing human GIST dataset with matched human serum was analyzed. Results: Unsupervised clustering revealed endothelial, smooth muscle, and tumor cells had high expression of KitL RNA in untreated tumors from KitV558Δ/+ mice. Imatinib therapy increased KitL RNA in all 3 cell types, suggesting that extra-tumoral KitL expression is dependent on oncogenic signaling (p Citation Format: Andrew D. Tieniber, Ferdinando Rossi, Andrew Hanna, Mengyuan Liu, Mark Etherington, Kevin Do, Laura Wang, Ronald P. DeMatteo. Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A017.

Introduction: MHC class I (MHC I) expression is necessary for the antitumoral lymphocyte response but its relationship to oncogene signaling is not well characterized. Gastrointestinal stromal tumor (GIST) is a common sarcoma caused by an activating Kit mutation and is sensitive to the specific Kit inhibitor imatinib. Using KitV558Δ/+ mice, a murine model of GIST, we found imatinib reduced MHC I expression on tumor cells, which we hypothesized was due to attenuation of type I interferon (IFN) signaling in the setting of oncogene inhibition. Methods: Using flow cytometry and Western blot, we studied tumoral MHC I expression in human GIST cell lines T1 and HG129 under conditions of IFN stimulation and imatinib inhibition. We applied the same methods in vivo in KitV558Δ/+ mice. To show that IFN signaling was responsible for tumoral MHC I expression, we used a blocking anti-IFNAR antibody to abrogate IFN signaling in KitV558Δ/+ mice. Finally, we validated our findings with RNA seq of 35 Kit exon 11 human GIST. Results: In human GIST cell lines, IFNα (1000U/mL) stimulated MHC I expression at 24h as measured on cell surface by mean florescence intensity (MFI) and by Western blot. This was accompanied by increased Stat1 and pStat1 protein. Imatinib (100nM) inhibited Stat1 activity and reduced MHC I expression by 12-34% on MFI, with and without IFNα stimulation. When Stat1 was knocked down with SiRNA, imatinib did not further decrease MHC I. In vivo, KitV558Δ/+ mice treated with imatinib had 50% reduction in tumoral MHC I expression MFI by flow (n=5/group). Concurrently, imatinib diminished mRNA expression of IFNb1 in bulk KitV558Δ/+ tumors by 8-fold. To demonstrate that IFN signaling contributed to GIST MHC I expression, we administered a blocking anti-IFNAR antibody in KitV558Δ/+ mice and found MHC I decreased by 34% on MFI, along with decreased pStat1 on western blot. In human GIST, imatinib significantly decreased MHC I gene expression on RNA seq (p = 0.03) and reduced IFNb1 mRNA by 77% (p = 0.04). Furthermore, there was significant correlation between MHC I genes and Stat1 expression (p< 0.0001). Taken together, this suggests that oncogene-dependent IFN signaling is responsible for MHC I expression in GIST. Conclusion: Kit oncogene inhibition reduced MHC I expression in GIST through decreased type I interferon production and signaling via Stat1. Thus, targeted therapies may reduce the antitumor immune response. Citation Format: Mengyuan Liu, Benjamin D Medina, Geraldo A Vitiello, Mark S Etherington, Nesteene J. Param, Mia E DiLolle, Timothy G. Bowler, Ferdinando Rossi, Ronald P. DeMatteo. Kit inhibition decreases tumoral MHC class I expression in gastrointestinal stromal tumors through reduction of type I interferon signaling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B27.

Perineuronal nets (PNNs) are aggregates of extracellular matrix molecules surrounding several types of neurons in the adult CNS, which contribute to stabilising neuronal connections. Interestingly, a reduction of PNN number and staining intensity has been observed in conditions associated with plasticity in the adult brain. However, it is not known whether spontaneous PNN changes are functional to plasticity and repair after injury. To address this issue, we investigated PNN expression in the vestibular nuclei of the adult mouse during vestibular compensation, namely the resolution of motor deficits resulting from a unilateral peripheral vestibular lesion. After unilateral labyrinthectomy, we found that PNN number and staining intensity were strongly attenuated in the lateral vestibular nucleus on both sides, in parallel with remodelling of excitatory and inhibitory afferents. Moreover, PNNs were completely restored when vestibular deficits of the mice were abated. Interestingly, in mice with genetically reduced PNNs, vestibular compensation was accelerated. Overall, these results strongly suggest that temporal tuning of PNN expression may be crucial for vestibular compensation.

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.

In the adult central nervous system (CNS) subsets of neurons are enwrapped by densely organized extracellular matrix structures, called perineuronal nets (PNNs). PNNs are formed at the end of critical periods and contribute to synapse stabilization. Enzymatic degradation of PNNs or genetic deletion of specific PNN components leads to the prolongation of the plasticity period. PNNs consist of extracellular matrix molecules, including chondroitin sulfate proteoglycans, hyaluronan, tenascins and link proteins. It has been recently shown that the chemorepulsive axon guidance protein semaphorin3A (Sema3A) is also a constituent of PNNs, binding with high affinity to the sugar chains of chondroitin sulfate proteoglycans. To elucidate whether the expression of Sema3A is modified in parallel with structural plasticity in the adult CNS, we examined Sema3A expression in the deep cerebellar nuclei of the adult mouse in a number of conditions associated with structural reorganization of the local connectivity. We found that Sema3A in PNNs is reduced during enhanced neuritic remodeling, in both physiological and injury-induced conditions. Moreover, we provide evidence that Sema3A is tightly associated with Purkinje axons and their terminals and its amount in the PNNs is related to Purkinje cell innervation of DCN neurons, but not to glutamatergic inputs. On the whole these data suggest that Sema3A may contribute to the growth-inhibitory properties of PNNs and Purkinje neurons may directly control their specific connection pattern through the release and capture of this guidance cue in the specialized ECM that surrounds their terminals.

The glial cells of the cerebellum, and particularly astrocytes and oligodendrocytes, are characterized by a remarkable phenotypic variety, in which highly peculiar morphological features are associated with specific functional features, unique among the glial cells of the entire CNS. Here, we provide a critical report about the present knowledge of the development of cerebellar glia, including lineage relationships between cerebellar neurons, astrocytes and oligodendrocytes, the origins and the genesis of the repertoire of glial types, and the processes underlying their acquisition of mature morphological and functional traits. In parallel, we describe and discuss some fundamental roles played by specific categories of glial cells during cerebellar development. In particular, we propose that Bergmann glia exerts a crucial scaffolding activity that, together with the organizing function of Purkinje cells, is necessary to achieve the normal pattern of foliation and layering of the cerebellar cortex. Moreover, we discuss some of the functional tasks of cerebellar astrocytes and oligodendrocytes that are distinctive of cerebellar glia throughout the CNS. Notably, we report about the regulation of synaptic signalling in the molecular and granular layer mediated by Bergmann glia and parenchymal astrocytes, and the functional interaction between oligodendrocyte precursor cells and neurons. On the whole, this review provides an extensive overview of the available literature and some novel insights about the origin and differentiation of the variety of cerebellar glial cells and their function in the developing and mature cerebellum.

Neural stem cells (NSCs) have become promising tools for basic research and regenerative medicine. Intracerebral transplantation studies have suggested that these cells may be able to adopt neuronal phenotypes typical of their engraftment site and to establish appropriate connections in the recipient circuitries. Here, we examined the in vivo neurogenic competence of well-characterized NSC lines subjected to in vitro priming and subsequent implantation into the adult intact mouse brain. Upon implantation into the hippocampus and, less frequently, in the striatum and in the cerebral cortex, numerous green fluorescent protein (GFP)-tagged cells acquired differentiated features indistinguishable from resident neurons. Upon closer examination, however, we found that this outcome resulted from fusion of donor cells with local neuronal elements generating long-term persistent GFP+ neuronal hybrids. This fusogenic behavior of NSCs was unexpected and also observed in coculture with E18 hippocampal immature neural cells, but not with microglia or astrocytes. Similar findings were consistently obtained with different NSC lines, mouse recipients, and donor cell-labeling methods. The frequent and cell type-specific fusion of donor NSCs with host neurons highlights a previously underestimated biological property of the nervous tissue that might prove profitable for basic and therapeutically oriented studies. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

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.

Recent studies on cortical processing of sensory information highlight the importance of multisensory integration, and define precise rules governing reciprocal influences between inputs of different sensory modalities. We propose that psychophysical interactions between different types of sensory stimuli and linguistic synaesthesia share common origins and mechanisms. To test this hypothesis, we compare neurophysiological findings with corpus-based analyses relating to linguistic synaesthesia. Namely, we present Williams’ hypothesis and its recent developments about the hierarchy of synaesthetic pairings, and examine critical aspects of this theory concerning universality, directionality, sensory categories, and usage of corpora. These theoretical issues are verified against linguistic data derived from corpus-based analyses of Italian synaesthetic pairings related to auditory and tactile modalities. Our findings reveal a strong parallel between linguistic synaesthesia and neurophysiological interactions between different sensory stimuli, suggesting that linguistic synaesthesia is affected by tendencies similar to the rules underlying the perceptual association of distinct sensory modalities.

During the past decades Neural Stem Cells have been considered as an alternative source of cells to replace lost neurons and NSC transplantation has been indicated as a promising treatment for neurodegenerative disorders. Nevertheless, the current understanding of NSC biology suggests that, far from being mere spare parts for cell replacement therapies, NSCs could play a key role in the pharmacology of neuroprotection and become protagonists of innovative treatments for neurodegenerative diseases. Here, we review this new emerging concept of NSC biology.

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.

CNS repair by cell transplantation requires new neurons to integrate into complex recipient networks. We assessed how the migratory route of transplanted granule neurons and the developmental stage of the host rat cerebellum influence engraftment. In both embryonic and postnatal hosts, granule cells can enter the cerebellar cortex and achieve correct placement along their natural migratory pathway. Donor neurons can also reach the internal granular layer from the white matter and integrate following an unusual developmental pattern. Although the frequency of correct positioning declines in parallel with cortical development, in mature recipients correct homing is more frequent through the unusual path. Following depletion of granule cell precursors in the host, more granule neurons engraft, but their ability for achieving correct placement is unchanged. Therefore, while the cerebellar environment remains receptive for granule cells even after the end of development, their full integration is partially hindered by the mature cortical architecture.

The subventricular zone (SVZ) of the lateral ventricle develops from residual progenitors of the embryonic lateral ganglionic eminence (LGE) and maintains neurogenic activity throughout life. Precursors from LGE/SVZ migrate to the olfactory bulb (OB) where they differentiate into local interneurons, principally in the granule layer and glomerular layer (GL). Byin situdye labeling, we show that neonatal and adult SVZ progenitors differentially contribute to neurochemically distinct types of periglomerular interneurons in the GL. Namely, calbindin-positive periglomerular cells are preferentially generated during early life, whereas calretinin- and tyrosine hydroxylase-expressing neurons are mainly produced at later ages. Furthermore, homochronic/heterochronic transplantation demonstrates that progenitor cells isolated from the LGE or SVZ at different stages (embryonic day 15 and postnatal days 2 and 30) engraft into the SVZ of neonatal or adult mice, migrate to the OB, and differentiate into local interneurons, including granule and periglomerular cells as well as other types of interneurons. The total number of integrated cells and the relative proportion of granule or periglomerular neurons change, according to the donor age, whereas they are weakly influenced by the recipient age. Analysis of the neurochemical phenotypes acquired by transplanted cells in the GL shows that donor cells of different ages also differentiate according to their origin, regardless of the host age. This suggests that progenitor cells at different ontogenetic stages are intrinsically directed toward specific lineages. Neurogenic processes occurring during development and in adult OB are not equivalent and produce different types of periglomerular interneurons as a consequence of intrinsic properties of the SVZ progenitors.

Regulation of neuritic growth is crucial for neural development, adaptation and repair. The intrinsic growth potential of nerve cells is determined by the activity of specific molecular sets, which sense environmental signals and sustain structural extension of neurites. The expression and function of these molecules are dynamically regulated by multiple mechanisms, which adjust the actual growth properties of each neuron population at different ontogenetic stages or in specific conditions. The neuronal potential for axon elongation and regeneration are restricted at the end of development by the concurrent action of several factors associated with the final maturation of neurons and of the surrounding tissue. In the adult, neuronal growth properties can be significantly modulated by injury, but they are also continuously tuned in everyday life to sustain physiological plasticity. Strict regulation of structural remodelling and neuritic elongation is thought to be required to maintain specific patterns of connectivity in the highly complex mammalian CNS. Accordingly, procedures that neutralize such mechanisms effectively boost axon growth in both intact and injured nervous system. Even in these conditions, however, aberrant connections are only formed in the presence of unusual external stimuli or experience. Therefore, growth regulatory mechanisms play an essentially permissive role by setting the responsiveness of neural circuits to environmental stimuli. The latter exert an instructive action and determine the actual shape of newly formed connections. In the light of this notion, efficient therapeutic interventions in the injured CNS should combine targeted manipulations of growth control mechanisms with task-specific training and rehabilitation paradigms.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits.

Gastrointestinal stromal tumor (GIST), originating from the interstitial cells of Cajal (ICC), is characterized by frequent activating mutations of the KIT receptor tyrosine kinase. Despite the clinical success of imatinib, which targets KIT, most patients with advanced GIST develop resistance and eventually die of the disease. The ETS family transcription factor ETV1 is a master regulator of the ICC lineage. Using mouse models of Kit activation and Etv1 ablation, we demonstrate that ETV1 is required for GIST initiation and proliferation in vivo, validating it as a therapeutic target. We further uncover a positive feedback circuit where MAP kinase activation downstream of KIT stabilizes the ETV1 protein, and ETV1 positively regulates KIT expression. Combined targeting of ETV1 stability by imatinib and MEK162 resulted in increased growth suppression in vitro and complete tumor regression in vivo. The combination strategy to target ETV1 may provide an effective therapeutic strategy in GIST clinical management. Significance: ETV1 is a lineage-specific oncogenic transcription factor required for the growth and survival of GIST. We describe a novel strategy of targeting ETV1 protein stability by the combination of MEK and KIT inhibitors that synergistically suppress tumor growth. This strategy has the potential to change first-line therapy in GIST clinical management. Cancer Discov; 5(3); 304–15. ©2015 AACR. See related commentary by Duensing, p. 231 This article is highlighted in the In This Issue feature, p. 213

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.

Repair of CNS injury or degeneration by cell replacement may lead to significant functional recovery only through faithful reconstruction of the original anatomical architecture. This is particularly relevant for point-to-point systems, where precisely patterned connections have to be re-established to regain adaptive function. Despite the major interest recently drawn on cell therapies, little is known about the mechanisms and the potentialities for specific integration of new neurons in the mature CNS. Major findings and concepts about this issue will be reviewed here, with special focus on work dealing with the Purkinje cell transplantation in the rodent cerebellum. These studies show that the adult CNS may provide some efficient information to direct cell engraftment and process outgrowth. On their side, immature cells may be able to induce adaptive changes in their adult partners to facilitate their incorporation in the recipient network. Despite the rather high degree of specific integration achieved in several different CNS regions, these processes are usually defective and long-distance connections are not rewired. Thus, although some potentialities for cell replacement exist in the mature CNS, full incorporation of new neurons in adult circuits is rarely observed. Indeed, intrinsic mechanisms for growth control as well as injury-induced changes in the properties and architecture of the nervous tissue contribute to hamper repair processes. As a consequence, crucial to obtain successful cell replacement and integration in the mature CNS is a deep understanding of the basic biological mechanisms that regulate the interactions between newly added elements and the recipient environment.

Cell replacement after neuronal degeneration in the adult CNS depends on the availability of specific cues to direct specification, differentiation and integration of newly born neurons into mature circuits. Following recent reports indicating that neurogenic signals may be reactivated in the adult injured CNS, here we asked whether such signals are expressed in the cerebellum after Purkinje cell degeneration. Thus, we compared the fate of embryonic cerebellar cells transplanted to the cerebella of adult wild-type and Purkinje cell degeneration (pcd) mutant mice. Donor cells were dissected from beta-actin-enhanced green fluorescent protein (EGFP) transgenic mice and transplanted as a single cell suspension. In both hosts, grafted cells generated all major cerebellar phenotypes, with a precise localization in the recipient cortex or white matter. Nevertheless, the phenotypic distributions showed striking quantitative differences. Most notably, in the pcd cerebellum there was a higher amount of Purkinje cells, while other phenotypes were less frequent. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrDU) incorporation revealed that in both hosts mitotic activity was strongly reduced shortly after transplantation, and virtually all donor Purkinje cells were actually generated before grafting. Together, these results indicate that some compensatory mechanisms operate in the pcd environment. However, the very low mitotic rate of transplanted cells suggests that the adult cerebellum, either wild-type or mutant, does not provide instructive neurogenic cues to direct the specification of uncommitted progenitors. Rather, specific replacement in mutant hosts is achieved through selective mechanisms that favour the survival and integration of donor Purkinje cells at the expense of other phenotypes.