UBE3A Regulates Synaptic Plasticity and Learning and Memory by Controlling SK2 Channel Endocytosis.

Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function.

ATM-dependent MiR-335 targets CtIP and modulates the DNA damage response.

ATM plays a critical role in cellular responses to DNA double-strand breaks (DSBs). We describe a new ATM-mediated DSB-induced DNA damage response pathway involving microRNA (miRNA): irradiation (IR)-induced DSBs activate ATM, which leads to the downregulation of miR-335, a miRNA that targets CtIP, which is an important trigger of DNA end resection in homologous recombination repair (HRR). We demonstrate that CREB is responsible for a large portion of miR-335 expression by binding to the promoter region of miR-335. CREB binding is greatly reduced after IR, corroborating with previous studies that IR-activated ATM phosphorylates CREB to reduce its transcription activity. Overexpression of miR-335 in HeLa cells resulted in reduced CtIP levels and post-IR colony survival and BRCA1 foci formation. Further, in two patient-derived lymphoblastoid cell lines with decreased post-IR colony survival, a "radiosensitive" phenotype, we demonstrated elevated miR-335 expression, reduced CtIP levels, and reduced BRCA1 foci formation. Colony survival, BRCA1 foci, and CtIP levels were partially rescued by miRNA antisense AMO-miR-335 treatment. Taken together, these findings strongly suggest that an ATM-dependent CREB-miR-335-CtIP axis influences the selection of HRR for repair of certain DSB lesions.

Rapid and progressive pulmonary fibrosis in 2 families with DNA repair deficiencies of undetermined etiology.

Known genetic causes of pediatric interstitial lung disease include disorders of surfactant metabolism, telomerase, and DNA repair. We report 4 children from 2 families with rapidly progressive and fatal pulmonary fibrosis. A novel DNA repair defect unrelated to the ataxia-telangiectasia mutated gene was found in 1 child from each family.

Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

OBJECTIVE: Previous reports of cells from patients with systemic lupus erythematosus (SLE) note that repair of single-strand breaks is delayed, and these lesions may be converted to double-strand breaks (DSBs) at DNA replication forks. We undertook this study to assess the integrity of DSB recognition, signaling, and repair mechanisms in B lymphoblastoid cell lines derived from patients with pediatric SLE. METHODS: Nine assays were used to interrogate DSB repair and recognition in lymphoblastoid cell lines from patients with pediatric SLE, including the neutral comet assay (NCA), colony survival assay (CSA), irradiation-induced foci formation for γ-H2AX and 53BP1 proteins, kinetics of phosphorylation of structural maintenance of chromosomes protein 1 (SMC1), postirradiation bromodeoxyuridine incorporation to evaluate S phase checkpoint integrity, monoubiquitination of Fanconi protein D2, ATM protein expression, and non-homologous DNA end joining protein expression and function. RESULTS: Three of the 9 assays revealed abnormal patterns of response to irradiation-induced DNA damage. The NCA and CSA yielded aberrant results in the majority of SLE lymphoblastoid cell lines. Abnormal prolongation of SMC1 phosphorylation was also noted in 2 of 16 SLE lymphoblastoid cell lines. CONCLUSION: Our data suggest that DSB repair is defective in some lymphoblastoid cell lines from pediatric patients with SLE, especially when assessed by both NCA and CSA. Since these studies are nonspecific, further studies of DNA repair and kinetics are indicated to further delineate the underlying pathogenesis of SLE and possibly identify therapeutic targets.

Functional characterization and targeted correction of ATM mutations identified in Japanese patients with ataxia-telangiectasia.

A recent challenge for investigators studying the progressive neurological disease ataxia-telangiectasia (A-T) is to identify mutations whose effects might be alleviated by mutation-targeted therapies. We studied ATM mutations in eight families of Japanese A-T patients (JPAT) and were able to identify all 16 mutations. The probands were compound heterozygotes in seven families, and one (JPAT2) was homozygous for a frameshift mutation. All mutations--four frameshift, two nonsense, four large genomic deletions, and six affecting splicing--were novel except for c.748C>T found in family JPAT6 and c.2639-384A>G found in family JPAT11/12. Using an established lymphoblastoid cell line (LCL) of patient JPAT11, ATM protein was restored to levels approaching wild type by exposure to an antisense morpholino oligonucleotide designed to correct a pseudoexon splicing mutation. In addition, in an LCL from patient JPAT8/9, a heterozygous carrier of a nonsense mutation, ATM levels could also be partially restored by exposure to readthrough compounds (RTCs): an aminoglycoside, G418, and a novel small molecule identified in our laboratory, RTC13. Taken together, our results suggest that screening and functional characterization of the various sorts of mutations affecting the ATM gene can lead to better identification of A-T patients who are most likely to benefit from rapidly developing mutation-targeted therapeutic technologies.

Assessing 'radiosensitivity' with kinetic profiles of γ-H2AX, 53BP1 and BRCA1 foci.

BACKGROUND AND PURPOSE: DNA repair assays to identify radiosensitive patients have had limited clinical implementation due to long turn-around times or limited specificity. This study evaluates γ-H2AX-Irradiation Induced Foci (IRIF) kinetics as a more rapid surrogate for the 'gold standard' colony survival assay (CSA) using several known DNA repair disorders as reference models. MATERIALS AND METHODS: Radiosensitive cells of known and unknown etiology were studied. γ-H2AX-IRIFs were quantified over 24 h, and the curves were fitted by combining logarithmic growth and exponential decay functions. Fitted values that differed from radionormal controls were considered aberrant and compared to CSA results. RESULTS: We observed 87% agreement of IRIF data with the CSA for the 14 samples tested. Analysis of γ-H2AX-IRIF kinetics for known repair disorders indicated similarities between an RNF168(-/-) cell line and an RS cell of unknown etiology. These cell lines were further characterized by a reduction in BRCA1-IRIF formation and G2/M checkpoint activation. CONCLUSIONS: γ-H2AX-IRIF kinetics showed high concordance with the CSA in RS populations demonstrating its potential as a more rapid surrogate assay. This method provides a means to globally identify defective DNA repair pathways in RS cells of unknown etiology through comparison with known DNA repair defects.