The genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1

Defective responses to DNA single strand breaks underlie various neurodegenerative diseases. However, the exact role of this repair pathway during the development and maintenance of the nervous system is unclear. Using murine neural-specific inactivation of Xrcc1, a factor that is critical for the repair of DNA single strand breaks, we found a profound neuropathology that is characterized by the loss of cerebellar interneurons. This cell loss was linked to p53-dependent cell cycle arrest and occurred as interneuron progenitors commenced differentiation. Loss of Xrcc1 also led to the persistence of DNA strand breaks throughout the nervous system and abnormal hippocampal function. Collectively, these data detail the in vivo link between DNA single strand break repair and neurogenesis and highlight the diverse consequences of specific types of genotoxic stress in the nervous system

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1

CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response

Aprataxin, defective in the neurodegenerative disorder ataxia oculomotor apraxia type 1, resolves abortive DNA ligation intermediates during DNA repair. Here, we demonstrate that aprataxin localizes at sites of DNA damage induced by high LET radiation and binds to mediator of DNA-damage checkpoint protein 1 (MDC1/NFBD1) through a phosphorylation-dependent interaction. This interaction is mediated via the aprataxin FHA domain and multiple casein kinase 2 di-phosphorylated S-D-T-D motifs in MDC1. X-ray structural and mutagenic analysis of aprataxin FHA domain, combined with modelling of the pSDpTD peptide interaction suggest an unusual FHA binding mechanism mediated by a cluster of basic residues at and around the canonical pT-docking site. Mutation of aprataxin FHA Arg29 prevented its interaction with MDC1 and recruitment to sites of DNA damage. These results indicate that aprataxin is involved not only in single strand break repair but also in the processing of a subset of double strand breaks presumably through its interaction with MDC1

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Disabled-1 alternative splicing in human fetal retina and neural tumors.

BACKGROUND: The Reelin-Dab1 signaling pathway plays a critical role in the positioning of migrating neurons, dendrite formation and lamination in the developing central nervous system. We have previously identified two alternatively spliced forms of Dab1 in the developing chick retina: an early form, Dab1-E, expressed in retinal progenitor cells, and a late form, Dab1 or Dab1-L, expressed in amacrine and ganglion cells. Compared to Dab1-L, Dab1-E lacks two exons that encode two Src family kinase (SFK) phosphorylation sites. PRINCIPAL FINDINGS: Both Dab1-L and Dab1-E-like transcripts were identified in human fetal retina. Expression of human Dab1-L in primary chick retinal cultures resulted in Reelin-mediated induction of SFK phosphorylation and formation of neurite-like processes. In contrast, human Dab1-E-expressing cells retained an undifferentiated morphology. The human Dab1 gene is located within a common fragile site, and it has been postulated that it may function as a tumor suppressor. Analysis of Dab1 splice forms in retinoblastoma and neuroblastoma tumor cells revealed relative enrichment of Dab1-L-like (includes exons 7 and 8) and Dab1-E-like (excludes exons 7 and 8) transcripts in retinoblastoma and neuroblastoma, respectively. Treatment of retinoblastoma cell line RB522A with Reelin resulted in increased tyrosine phosphorylation of Dab1. As Nova2 has previously been implicated in the exclusion of exons 9B and 9C in Dab1, we examined the expression of this splicing factor in neuroblastoma and retinoblastoma cell lines. Nova2 was only detected in neuroblastoma cells, suggesting a correlation between Nova2 expression and increased levels of Dab1-E-like splice forms in neuroblastoma. CONCLUSIONS: These results indicate that alternative splicing of Dab1 is conserved in avian and mammalian species, with Dab1-L driving SFK phosphorylation in both species. Dab1-E- and Dab-L-like isoforms are also expressed in childhood neural tumors, with preferential enrichment of Dab1-L-like and Dab1-E-like isoforms in retinoblastoma and neuroblastoma, respectively

Dynamic Nature of Cleavage Bodies and Their Spatial Relationship to DDX1 Bodies, Cajal Bodies, and Gems

DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature

Radiographic and perioperative outcomes following anterior thoracic vertebral body tethering and posterior lumbar spine tethering: a pilot series.

BACKGROUND AND CONTEXT:  In patients with adolescent idiopathic scoliosis (AIS) of main thoracic and lumbar spine regions, combined anterior thoracic vertebral body tethering and posterior lumbar spine tethering (ATVBT/PLST) is a novel non-fusion treatment option for growth modulation and conservation of motion. METHODS:  Fourteen patients with AIS who underwent ATVBT/PLST with at least 2-year follow-up were included. Primary outcomes included quality of life as assessed by SRS-22 instruments, radiographic analysis, and revision operations. We secondarily reported perioperative metrics and post-operative opiate morphine equivalents (OME). Clinical success was defined as patients who achieved skeletal maturity with ≤ 30° curve magnitude of both their main thoracic and thoracolumbar/lumbar curves and who did not undergo posterior spine instrumentation and fusion (PSIF). RESULTS: Patients had a mean age of 11.6 years (range 10-14 years), majority were girls (92%), and mean follow-up was 3.0 years (range 2-4.8 years). All patients were skeletally immature with a Risser ≤ 2. Included curves were Lenke 1C, 3C, or 6C. Mean preoperative curve magnitudes were 53° ± 8° (range 45°-65°) main thoracic and 49° ± 9° (range 40°-62°) thoracolumbar/lumbar curves. At most recent follow-up, patients had a mean main thoracic curve of 29° ± 8° (range 15°-40°) and a mean thoracolumbar/lumbar curve of 20° ± 15° (range 4°-35°). 50% required a revision operation. Cable breakage occurred in 43%, which did not always require revision. One patient progressed to thoracic fusion, but no patient underwent lumbar fusion. Patients had a mean SRS-22 outcome score of 4.2 ± 0.4. CONCLUSIONS: ATVBT/PLST is a potential alternative to spine fusion for select immature patients with AIS at a minimum 2-year follow-up. ATVBT/PLST potentially offers motion conservation at the cost of a higher revision rate. Further study and reporting of results are necessary to refine indications and techniques, which in turn will improve outcomes of this procedure. LEVEL OF EVIDENCE: Level IV-Case series without comparative group