NOGO Receptor 1 (RTN4R) regulates axonal growth, as well as axon regeneration after injury. The gene maps to the 22q11.2 schizophrenia susceptibility locus and is thus a strong functional and positional candidate gene.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) cleaves the phosphodiester bond between a covalently stalled topoisomerase I (Topo I) and the 3' end of DNA. Stalling of Topo I at DNA strand breaks is induced by endogenous DNA damage and the Topo I-specific anticancer drug camptothecin (CPT). The H493R mutation of Tdp1 causes the neurodegenerative disorder spinocerebellar ataxia with axonal neuropathy (SCAN1). Contrary to the hypothesis that SCAN1 arises from catalytically inactive Tdp1, Tdp1-/- mice are indistinguishable from wild-type mice, physically, histologically, behaviorally, and electrophysiologically. However, compared to wild-type mice, Tdp1-/- mice are hypersensitive to CPT and bleomycin but not to etoposide. Consistent with earlier in vitro studies, we show that the H493R Tdp1 mutant protein retains residual activity and becomes covalently trapped on the DNA after CPT treatment of SCAN1 cells. This result provides a direct demonstration that Tdp1 repairs Topo I covalent lesions in vivo and suggests that SCAN1 arises from the recessive neomorphic mutation H493R. This is a novel mechanism for disease since neomorphic mutations are generally dominant.

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

Alternative transcription start site (TSS) usage plays important roles in transcriptional control of mammalian gene expression. The growing interest in alternative TSSs and their role in genome diversification spawned many single-gene studies on differential usages of tissue-specific or temporal-specific alternative TSSs. However, exploration of the switching usage of alternative TSS usage on a genomic level, especially in the central nervous system, is largely lacking.

Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.

We have identified active enhancers in the mouse cerebellum at embryonic and postnatal stages which provides a view of novel enhancers active during cerebellar development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Furthermore, by comparing our data with relevant brain development ENCODE histone profiles and cerebellar single-cell datasets we have been able to generalize and expand on the presented analyses, respectively. We have made the results of our analyses available online in the Developing Mouse Cerebellum Enhancer Atlas, where our dataset can be efficiently queried, curated and exported by the scientific community to facilitate future research efforts. Our study provides a valuable resource for studying the dynamics of gene expression regulation by enhancers in the developing cerebellum and delivers a rich dataset of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.