Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice.

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.

Tonic activation of Bax primes neural progenitors for rapid apoptosis through a mechanism preserved in medulloblastoma.

Commitment to survival or apoptosis within expanding progenitor populations poses distinct risks and benefits to the organism. We investigated whether specialized mechanisms regulate apoptosis in mouse neural progenitors and in the progenitor-derived brain tumor medulloblastoma. Here, we identified constitutive activation of proapoptotic Bax, maintained in check by Bcl-xL, as a mechanism for rapid cell death, common to postnatal neural progenitors and medulloblastoma. We found that tonic activation of Bax in cerebellar progenitors, along with sensitivity to DNA damage, was linked to differentiation state. In cerebellar progenitors, active Bax localized to mitochondria, where it was bound to Bcl-xL. Disruption of Bax:Bcl-xL binding by BH3-mimetic ABT 737 caused rapid apoptosis of cerebellar progenitors and primary murine medulloblastoma cells. Conditional deletion of Mcl-1, in contrast, did not cause death of cerebellar progenitors. Our findings identify a mechanism for the sensitivity of brain progenitors to typical anticancer therapies and reveal that this mechanism persists in medulloblastoma, a malignant brain tumor markedly sensitive to radiation and chemotherapy.

An Adeno-Associated Virus-Based Toolkit for Preferential Targeting and Manipulating Quiescent Neural Stem Cells in the Adult Hippocampus.

Quiescent neural stem cells (qNSCs) with radial morphology are the only proven source of new neurons in the adult mammalian brain. Our understanding of the roles of newly generated neurons depends on the ability to target and manipulate adult qNSCs. Although various strategies have been developed to target and manipulate adult hippocampal qNSCs, they often suffer from prolonged breeding, low recombination efficiency, and non-specific labeling. Therefore, developing a readily manufactured viral vector that allows flexible packaging and robust expression of various transgenes in qNSCs is a pressing need. Here, we report a recombinant adeno-associated virus serotype 4 (rAAV4)-based toolkit that preferentially targets hippocampal qNSCs and allows for lineage tracing, functional analyses, and activity manipulation of adult qNSCs. Importantly, targeting qNSCs in a non-Cre-dependent fashion opens the possibility for studying qNSCs in less genetically tractable animal species and may have translational impact in gene therapy by preferentially targeting qNSCs.

Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum.

Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

Radiation Sensitivity in a Preclinical Mouse Model of Medulloblastoma Relies on the Function of the Intrinsic Apoptotic Pathway.

While treatments that induce DNA damage are commonly used as anticancer therapies, the mechanisms through which DNA damage produces a therapeutic response are incompletely understood. Here we have tested whether medulloblastomas must be competent for apoptosis to be sensitive to radiotherapy. Whether apoptosis is required for radiation sensitivity has been controversial. Medulloblastoma, the most common malignant brain tumor in children, is a biologically heterogeneous set of tumors typically sensitive to radiation and chemotherapy; 80% of medulloblastoma patients survive long-term after treatment. We used functional genetic studies to determine whether the intrinsic apoptotic pathway is required for radiation to produce a therapeutic response in mice with primary, Shh-driven medulloblastoma. We found that cranial radiation extended the survival of medulloblastoma-bearing mice and induced widespread apoptosis. Expression analysis and conditional deletion studies showed that Trp53 (p53) was the predominant transcriptional regulator activated by radiation and was strictly required for treatment response. Deletion of Bax, which blocked apoptosis downstream of p53, was sufficient to render tumors radiation resistant. In apoptosis-incompetent, Bax-deleted tumors, radiation activated p53-dependent transcription without provoking cell death and caused two discrete populations to emerge. Most radiated tumor cells underwent terminal differentiation. Perivascular cells, however, quickly resumed proliferation despite p53 activation, behaved as stem cells, and rapidly drove recurrence. These data show that radiation must induce apoptosis in tumor stem cells to be effective. Mutations that disable the intrinsic apoptotic pathways are sufficient to impart radiation resistance. We suggest that medulloblastomas are typically sensitive to DNA-damaging therapies, because they retain apoptosis competence. Cancer Res; 76(11); 3211-23. ©2016 AACR.

Activity-dependent signaling mechanisms regulating adult hippocampal neural stem cells and their progeny.

Adult neural stem cells (NSCs) reside in a restricted microenvironment, where their development is controlled by subtle and presently underexplored cues. This raises a significant question: what instructions must be provided by this supporting niche to regulate NSC development and functions? Signaling from the niche is proposed to control many aspects of NSC behavior, including balancing the quiescence and proliferation of NSCs, determining the cell division mode (symmetric versus asymmetric), and preventing premature depletion of stem cells to maintain neurogenesis throughout life. Interactions between neurogenic niches and NSCs also govern the homeostatic regulation of adult neurogenesis under diverse physiological, environmental, and pathological conditions. An important implication from revisiting many previously-identifi ed regulatory factors is that most of them (e.g., the antidepressant fluoxetine and exercise) affect gross neurogenesis by acting downstream of NSCs at the level of intermediate progenitors and neuroblasts, while leaving the NSC pool unaffected. Therefore, it is critically important to address how various niche components, signaling pathways, and environmental stimuli differentially regulate distinct stages of adult neurogenesis.

Hexokinase-2-mediated aerobic glycolysis is integral to cerebellar neurogenesis and pathogenesis of medulloblastoma.

BACKGROUND: While aerobic glycolysis is linked to unconstrained proliferation in cancer, less is known about its physiological role. Why this metabolic program that promotes tumor growth is preserved in the genome has thus been unresolved. We tested the hypothesis that aerobic glycolysis derives from developmental processes that regulate rapid proliferation. METHODS: We performed an integrated analysis of metabolism and gene expression in cerebellar granule neuron progenitors (CGNPs) with and without Sonic Hedgehog (Shh), their endogenous mitogen. Because our analysis highlighted Hexokinase-2 (Hk2) as a key metabolic regulator induced by Shh, we studied the effect of conditional genetic Hk2 deletion in CGNP development. We then crossed Hk2 conditional knockout mice with transgenic SmoM2 mice that develop spontaneous medulloblastoma and determined changes in SmoM2-driven tumorigenesis. RESULTS: We show that Shh and phosphoinositide 3-kinase (PI3K) signaling combine to induce an Hk2-dependent glycolytic phenotype in CGNPs. This phenotype is recapitulated in medulloblastoma, a malignant tumor of CGNP origin. Importantly, cre-mediated ablation of Hk2 abrogated aerobic glycolysis, disrupting CGNP development and Smoothened-induced tumorigenesis. Comparing tumorigenesis in medulloblastoma-prone SmoM2 mice with and without functional Hk2, we demonstrate that loss of aerobic glycolysis reduces the aggressiveness of medulloblastoma, causing tumors to grow as indolent lesions and allowing long-term survival of tumor bearing mice. CONCLUSIONS: Our investigations demonstrate that aerobic glycolysis in cancer derives from developmental mechanisms that persist in tumorigenesis. Moreover, we demonstrate in a primary tumor model the anti-cancer potential of blocking aerobic glycolysis by targeting Hk2.See commentary article:http://www.biomedcentral.com/1741-7007/11/3.