Neurons in the superior olivary complex (SOC) integrate excitatory and inhibitory inputs to localize sounds in space. The majority of these inhibitory inputs have been thought to arise within the SOC from the medial nucleus of the trapezoid body (MNTB). However, recent work demonstrates that glycinergic innervation of the SOC persists in Egr2; En1(CKO) mice that lack MNTB neurons, suggesting that there are other sources of this innervation (Jalabi et al., 2013). To study the development of MNTB- and non-MNTB-derived glycinergic SOC innervation, we compared immunostaining patterns of glycine transporter 2 (GlyT2) at several postnatal ages in control and Egr2; En1(CKO) mice. GlyT2 immunostaining was present at birth (P0) in controls and reached adult levels by P7 in the superior paraolivary nucleus (SPN) and by P12 in the lateral superior olive (LSO). In Egr2; En1(CKO) mice, glycinergic innervation of the LSO developed at a similar rate but was delayed by one week in the SPN. Conversely, consistent reductions in the number of GlyT2(+) boutons located on LSO somata were seen at all ages in Egr2; En1(CKO) mice, while these numbers reached control levels in the SPN by adulthood. Dendritic localization of GlyT2+ boutons was unaltered in both the LSO and SPN of adult Egr2; En1(CKO) mice. On the postsynaptic side, adult Egr2; En1(CKO) mice had reduced glycine receptor α1 (GlyRα1) expression in the LSO but normal levels in the SPN. GlyRα2 was not expressed by LSO or SPN neurons in either genotype. These findings contribute important information for understanding the development of MNTB- and non-MNTB-derived glycinergic pathways to the mouse SOC.

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive, malignant tumors and are the most common malignant brain tumor in children under 6 months of age. Currently, there is no standard treatment for AT/RT. Recent studies have reported potential anti-tumoral properties of ribavirin, a guanosine analog and anti-viral molecule approved by the Food and Drug Administration for treatment of hepatitis C. We previously demonstrated that ribavirin inhibited glioma cell growth in vitro and in vivo. Based on these results and the fact that no pre-clinical model of ribavirin in AT/RT exists, we decided to investigate the effect of ribavirin on several human AT/RT cell lines (BT12, BT16, and BT37) both in vitro and in vivo. We provide evidence that ribavirin has a significant impact on AT/RT cell growth and increases cell cycle arrest and cell death, potentially through modulation of the eIF4E and/or EZH2 pathways. Interestingly, using scratch wound and transwell Boyden chamber assays, we observed that ribavirin also impairs AT/RT cell migration, invasion, and adhesion. Finally, we demonstrate that ribavirin significantly improves the survival of mice orthotopically implanted with BT12 cells. Our work establishes that ribavirin is effective against AT/RT by decreasing tumoral cell growth and dissemination and could represent a new therapeutic option for children with this deadly disease.

Clinical trials involving anti-programmed cell death protein-1 (anti-PD-1) failed to demonstrate improved overall survival in glioblastoma (GBM) patients. This may be due to the expression of alternative checkpoints such as B- and T- lymphocyte attenuator (BTLA) on several immune cell types including regulatory T cells. Murine GBM models indicate that there is significant upregulation of BTLA in the tumor microenvironment (TME) with associated T cell exhaustion. We investigate the use of antibodies against BTLA and PD-1 on reversing immunosuppression and increasing long-term survival in a murine GBM model. C57BL/6 J mice were implanted with the murine glioma cell line GL261 and randomized into 4 arms: (i) control, (ii) anti-PD-1, (iii) anti-BTLA, and (iv) anti-PD-1 + anti-BTLA. Kaplan-Meier curves were generated for all arms. Flow cytometric analysis of blood and brains were done on days 11 and 16 post-tumor implantation. Tumor-bearing mice treated with a combination of anti-PD-1 and anti-BTLA therapy experienced improved overall long-term survival (60%) compared to anti-PD-1 (20%) or anti-BTLA (0%) alone (P = .003). Compared to monotherapy with anti-PD-1, mice treated with combination therapy also demonstrated increased expression of CD4+ IFN-γ (P < .0001) and CD8+ IFN-γ (P = .0365), as well as decreased levels of CD4+ FoxP3+ regulatory T cells on day 16 in the brain (P = .0136). This is the first preclinical investigation into the effects of combination checkpoint blockade with anti-PD-1 and anti-BTLA treatment in GBM. We also show a direct effect on activated immune cell populations such as CD4+ and CD8 + T cells and immunosuppressive regulatory T cells through this combination therapy.

Olfactory neuroblastoma (ONB) is a rare malignant neoplasm arising in the upper portion of the sinonasal cavity. To better understand the genetic bases for ONB, here we perform whole exome and whole genome sequencing as well as single nucleotide polymorphism array analyses in a series of ONB patient samples. Deletions involving the dystrophin (DMD) locus are found in 12 of 14 (86%) tumors. Interestingly, one of the remaining tumors has a deletion in LAMA2, bringing the number of ONBs with deletions of genes involved in the development of muscular dystrophies to 13 or 93%. This high prevalence implicates an unexpected functional role for genes causing hereditary muscular dystrophies in ONB.

Medulloblastoma (MB) is the most common brain malignancy in children, and is still responsible for significant mortality and morbidity. The aim of this study was to assess the safety and efficacy of Disulfiram (DSF), an FDA-approved inhibitor of Aldehyde-Dehydrogenase (ALDH), and Copper (Cu++) in human SSH-driven and Group 3 MB. The molecular mechanisms, effect on cancer-stem-cells (CSC) and DNA damage were investigated in xenograft models.

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.

Little is known about the genetic pathways and transcription factors that control development and maturation of central auditory neurons. En1, a gene expressed by a subset of developing and mature superior olivary complex (SOC) cells, encodes a homeodomain transcription factor important for neuronal development in the midbrain, cerebellum, hindbrain and spinal cord. Using genetic fate-mapping techniques, we show that all En1-lineal cells in the SOC are neurons and that these neurons are glycinergic, cholinergic and GABAergic in neurotransmitter phenotype. En1 deletion does not interfere with specification or neural fate of these cells, but does cause aberrant positioning and subsequent death of all En1-lineal SOC neurons by early postnatal ages. En1-null cells also fail to express the transcription factor FoxP1, suggesting that FoxP1 lies downstream of En1. Our data define important roles for En1 in the development and maturation of a diverse group of brainstem auditory neurons.

Mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, an autosomal recessive neurodegenerative disease presenting with severe levodopa-responsive parkinsonism and pyramidal disturbances. Understanding the PARK15 pathogenesis might thus provide clues on the mechanisms of maintenance of brain dopaminergic neurons, the same which are lost in Parkinson's disease. The protein(s) encoded by FBXO7 remain very poorly characterized. Here, we show that two protein isoforms are expressed from the FBXO7 gene in normal human cells. The isoform 1 is more abundant, particularly in primary skin fibroblasts. Both isoforms are undetectable in cell lines from the PARK15 patient of an Italian family; the isoform 1 is undetectable and the isoform 2 is severely decreased in the patients from a Dutch PARK15 family. In human cell lines and mouse primary neurons, the endogenous or over-expressed, wild type FBXO7 isoform 1 displays mostly a diffuse nuclear localization. An intact N-terminus is needed for the nuclear FBXO7 localization, as N-terminal modification by PARK15-linked missense mutation, or N-terminus tag leads to cytoplasmic mislocalization. Furthermore, the N-terminus of wild type FBXO7 (but not of mutant FBXO7) is able to confer nuclear localization to profilin (a cytoplasmic protein). Our data also suggest that overexpressed mutant FBXO7 proteins (T22M, R378G and R498X) have decreased stability compared to their wild type counterpart. In human brain, FBXO7 immunoreactivity was highest in the nuclei of neurons throughout the cerebral cortex, intermediate in the globus pallidum and the substantia nigra, and lowest in the hippocampus and cerebellum. In conclusion, the common cellular abnormality found in the PARK15 patients from the Dutch and Italian families is the depletion of the FBXO7 isoform 1, which normally localizes in the cell nucleus. The activity of FBXO7 in the nucleus appears therefore crucial for the maintenance of brain neurons and the pathogenesis of PARK15.

ACT001, which is derived from an ancient anti-inflammatory drug, has been shown to cross the blood-brain barrier in preclinical studies and has demonstrated anti-glioblastoma (GBM) activity in clinical trials. However, its pharmacological potential for anti-GBM immune response modulation remains unclear. The chemical structure of ACT001 indicates that it may bind to STAT3 and thus modulate antitumor immune response. Methods: Bioinformatics and immunohistochemistry (IHC) were used to assess STAT3 and PD-L1 expression in gliomas. Western blotting, RT-PCR and immunofluorescence were used to detect PD-L1 and p-STAT3 expression in glioma cells exposed to ACT001. Chromatin immunoprecipitation, an ACT001-Biotin probe, and a dual-luciferase reporter assay were used to detect direct modulation. The in vivo efficacy of ACT001 was evaluated in GL261 murine glioma model. Survival analyses were conducted using the log-rank (Mantel-Cox) test. Results: Bioinformatic analysis of 1,837 samples from 4 public glioma datasets showed that STAT3 mRNA expression was correlated with the degree of malignancy and therapeutic resistance and that STAT3 mRNA expression was related to immunosuppression, leukocyte infiltration, and PD-L1 expression. IHC staining of 53 tissue samples confirmed that relatively high phosphorylated STAT3 and PD-L1 protein expression was associated with a relatively advanced World Health Organization (WHO) glioma grade. Next, we confirmed that ACT001 treatment reduced PD-L1 expression and STAT3 phosphorylation. An ACT001-biotin probe was used to verify that ACT001 bound to STAT3. We also demonstrated that STAT3 bound to the PD-L1 promoter. The inhibition of PD-L1 expression and STAT3 phosphorylation by ACT001 could be rescued by STAT3 overexpression. Additionally, ACT001 inhibited GBM growth and decreased PD-L1 expression in vivo. The expression of the M2 markers CD206 and CD163 was decreased, while that of the antitumor immune markers iNOS and IFNγ was increased by ACT001 in vivo. Conclusion: Our results demonstrate that STAT3 plays a key role in immunosuppression of glioma and is inhibited by ACT001. ACT001 inhibits PD-L1 transcription and modulates anti-tumor immune response in glioma bearing mice. These findings will help us to understand the mechanism of ACT001 in GBM therapy.

The roles of T (brachyury) isoforms in chordomas remain unclear. This study aimed to investigate the different roles and mechanisms of them in chordomas.