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Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy,many patients succumb to the disease,and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro,in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB. View Publication

While no effective therapy is available for the treatment of methamphetamine (METH)-induced neurotoxicity,aerobic exercise is being proposed to improve depressive symptoms and substance abuse outcomes. The present study focuses on the effect of exercise on METH-induced aberrant neurogenesis in the hippocampal dentate gyrus in the context of the blood-brain barrier (BBB) pathology. Mice were administered with METH or saline by i.p. injections for 5 days with an escalating dose regimen. One set of mice was sacrificed 24 h post last injection of METH,and the remaining animals were either subjected to voluntary wheel running (exercised mice) or remained in sedentary housing (sedentary mice). METH administration decreased expression of tight junction (TJ) proteins and increased BBB permeability in the hippocampus. These changes were preserved post METH administration in sedentary mice and were associated with the development of significant aberrations of neural differentiation. Exercise protected against these effects by enhancing the protein expression of TJ proteins,stabilizing the BBB integrity,and enhancing the neural differentiation. In addition,exercise protected against METH-induced systemic increase in inflammatory cytokine levels. These results suggest that exercise can attenuate METH-induced neurotoxicity by protecting against the BBB disruption and related microenvironmental changes in the hippocampus. View Publication

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases,for example,in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency,which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions,deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template,such as an introduced single-stranded oligo DNA nucleotide (ssODN),allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ,editing by HDR remains inefficient and can be corrupted by additional indels,preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore,targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations,and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB,we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation,whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach,we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons,which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9,facilitating study of human disease. View Publication

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation,but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358,normally added late in NPC biogenesis. SUN1,a torsinA-related molecule implicated in interphase NPC biogenesis,also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice,NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology. View Publication

The generation of differentiated and functional neurons is a complex process,which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200,miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells,induction of miR-200 family was observed in differentiating neural stem cells,demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However,inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells,an inverse relationship was observed between expression of reprogramming transcription factors (SOX2,KLF4,NANOG,OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover,we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion,our results indicate that increased expression of the miR-200 family promotes neuronal differentiation,while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4. View Publication

BACKGROUND Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However,resistance inevitably emerges against these therapies,particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels,p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer,neurosphere,and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics,100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS Daoy,ONS76,UW228,and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth,induced apoptosis,and sensitized cells to SHH agents. Notably,RSK expression is correlated with SHH patients. In mice,BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly,BI-D1870 crosses the BBB,acting as a scaffold for development of more long-lived RSK inhibitors. View Publication

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD,and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs,using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected,cell lines harboring the PSEN2 N141I mutation displayed an increase in the A$\beta$42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit,restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased A$\beta$42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in A$\beta$42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology. View Publication

Microglia positively affect neural progenitor cell physiology through the release of inflammatory mediators or trophic factors. We demonstrated previously that reactive microglia foster K(ATP) -channel expression and that blocking this channel using glibenclamide administration enhances striatal neurogenesis after stroke. In this study,we investigated whether the microglial K(ATP) -channel directly influences the activation of neural precursor cells (NPCs) from the subventricular zone using transgenic Csf1r-GFP mice. In vitro exposure of NPCs to lipopolysaccharide and interferon-gamma resulted in a significant decrease in precursor cell number. The complete removal of microglia from the culture or exposure to enriched microglia culture also decreased the precursor cell number. The addition of glibenclamide rescued the negative effects of enriched microglia on neurosphere formation and promoted a 20% improvement in precursor cell number. Similar results were found using microglial-conditioned media from isolated microglia. Using primary mixed glial and pure microglial cultures,glibenclamide specifically targeted reactive microglia to restore neurogenesis and increased the microglial production of the chemokine monocyte chemoattractant protein-1 (MCP-1). These findings provide the first direct evidence that the microglial K(ATP) -channel is a regulator of the proliferation of NPCs under inflammatory conditions. View Publication

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs),cancers occurring increasingly in children to young adults,is poorly understood. We present a transplantable tumour line,maintained in immune-compromised mice,and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays,spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model,with parallel studies on 27 primary hFL-HCC tumours,provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple,normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. View Publication

New therapeutic strategies are needed in the treatment of asthma besides vaccines and pharmacotherapies. For the development of novel therapies,the use of mesenchymal stem cells (MSCs) is a promising approach in regenerative medicine. Delivery of compact bone (CB) derived MSCs to the injured lungs is an alternative treatment strategy for chronic asthma. In this study,we aimed to isolate highly enriched population of MSCs from mouse CB with regenerative capacity,and to investigate the impact of these cells in airway remodeling and inflammation in experimental ovalbumin-induced mouse model of chronic asthma. mCB-MSCs were isolated,characterized,labeled with GFP and then transferred into mice with chronic asthma developed by ovalbumin (OVA) provocation. Histopathological changes including basement membrane,epithelium,subepithelial smooth thickness and goblet cell hyperplasia,and MSCs migration to lung tissues were evaluated. These histopathological alterations were increased in ovalbumin-treated mice compared to PBS group (P<0.001). Intravenous administration of mCB-MSC significantly reduced these histopathological changes in both distal and proximal airways (P<0.001). We showed that GFP-labeled MSCs were located in the lungs of OVA group 2weeks after intravenous induction. mCB-MSCs also significantly promoted Treg response in ovalbumin-treated mice (OVA+MSC group) (P<0.037). Our studies revealed that mCB-MSCs migrated to lung tissue and suppressed histopathological changes in murine model of asthma. The results reported here provided evidence that mCB-MSCs may be an alternative strategy for the treatment of remodeling and inflammation associated with chronic asthma. View Publication