Structure-based prediction and characterization of photo-crosslinking in native protein-RNA complexes.

UV-crosslinking of protein and RNA in direct contacts has been widely used to study protein-RNA complexes while our understanding of the photo-crosslinking mechanisms remains poor. This knowledge gap is due to the challenge of precisely mapping the crosslink sites in protein and RNA simultaneously in their native sequence and structural contexts. Here we systematically analyze protein-RNA interactions and photo-crosslinking by bridging crosslinked nucleotides and amino acids mapped using different assays with protein-RNA complex structures. We developed a computational method PxR3D-map which reliably predicts crosslink sites using structural information characterizing protein-RNA interaction interfaces. Analysis of the informative features revealed that photo-crosslinking is facilitated by base stacking with not only aromatic residues, but also dipeptide bonds that involve glycine, and distinct mechanisms are utilized by different RNA-binding domains. Our work suggests protein-RNA photo-crosslinking is highly selective in the cellular environment, which can guide data interpretation and further technology development for UV-crosslinking-based assays.

Characterising collateral vessels in eyes with branch retinal vein occlusions using widefield swept-source optical coherence tomography angiography.

BACKGROUND/AIMS: To characterise the morphology, location and functional significance of both macular and extramacular collateral vessels (CVs) in patients with a history of branch retinal vein occlusion (BRVO) using widefield swept-source optical coherence tomography angiography (WF SS OCTA). METHODS: Patients with a history of BRVO underwent WF SS OCTA testing to acquire 12×12 mm images, which were evaluated for CVs and non-perfusion area (NPA). Region of interest analysis of individual CVs was performed to identify correlations between CV size, depth and retinal location. Mixed effects multivariate regression analyses of factors associated with NPA and visual acuity (VA) were performed. RESULTS: Fifty-five CVs were identified in 28 BRVO eyes from 27 patients. CVs were identified in 42.9% (12/28) of eyes with a history of BRVO, and of these, 45.5% (25/55) were extramacular. The majority of CVs (87.3%, 48/55) coursed through both the superficial and the deep capillary plexus (DCP), while a subset (12.7%, 7/55) were strictly superficial. No CVs were found to course strictly through the DCP alone. CV depth increased with distance from the optic disc (p=0.011) and CV size increased with distance from the fovea (p=0.005). There were no statistically significant associations between CVs and NPA, or between CVs and VA. CONCLUSIONS: WF SS OCTA revealed that a large fraction of CVs that form after BRVO are extramacular, and the morphology of CVs varies as a function of retinal location. Depth-resolved study of CVs may offer valuable insights on the pathophysiological mechanisms leading to the development of macular oedema.

The Loss of TBK1 Kinase Activity in Motor Neurons or in All Cell Types Differentially Impacts ALS Disease Progression in SOD1 Mice.

DNA sequence variants in the TBK1 gene associate with or cause sporadic or familial amyotrophic lateral sclerosis (ALS). Here we show that mice bearing human ALS-associated TBK1 missense loss-of-function mutations, or mice in which the Tbk1 gene is selectively deleted in motor neurons, do not display a neurodegenerative disease phenotype. However, loss of TBK1 function in motor neurons of the SOD1G93A mouse model of ALS impairs autophagy, increases SOD1 aggregation, and accelerates early disease onset without affecting lifespan. By contrast, point mutations that decrease TBK1 kinase activity in all cells also accelerate disease onset but extend the lifespan of SOD1 mice. This difference correlates with the failure to activate high levels of expression of interferon-inducible genes in glia. We conclude that loss of TBK1 kinase activity impacts ALS disease progression through distinct pathways in different spinal cord cell types and further implicate the importance of glia in neurodegeneration.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS.

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non-cell-autonomous manner to promote disease progression.

Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-α gene expression.

Extraordinary single-cell diversity is generated in the vertebrate nervous system by the combinatorial expression of the clustered protocadherin genes (Pcdhα, -ß, and -γ). This diversity is generated by a combination of stochastic promoter choice and alternative pre-mRNA splicing. Here we show that both the insulator-binding protein CTCF and the cohesin complex subunit Rad21 bind to two highly conserved DNA sequences, the first within and the second downstream of transcriptionally active Pcdhα promoters. Both CTCF and Rad21 bind to these sites in vitro and in vivo, this binding directly correlates with alternative isoform expression, and knocking down CTCF expression reduces alternative isoform expression. Remarkably, a similarly spaced pair of CTCF/Rad21 binding sites was identified within a distant enhancer element (HS5-1), which is required for normal levels of alternative isoform expression. We also identify an additional, unique regulatory role for cohesin, as Rad21 binds to another enhancer (HS7) independently of CTCF, and knockdown of Rad21 reduces expression of the constitutive, biallelically expressed Pcdhα isoforms αc1 and αc2. We propose that CTCF and the cohesin complex initiate and maintain Pcdhα promoter choice by mediating interactions between Pcdhα promoters and enhancers.

Mouse model predicts effects of smoking and varenicline on event-related potentials in humans.

BACKGROUND: Nicotine alters auditory event-related potentials (ERPs) in rodents and humans and is an effective treatment for smoking cessation. Less is known about the effects of the partial nicotine agonist varenicline on ERPs. METHODS: We measured the effects of varenicline and nicotine on the mouse P20 and varenicline and smoking on the human P50 in a paired-click task. Eighteen mice were tested following nicotine, varenicline, and their combination. One hundred and fourteen current smokers enrolled in a placebo-controlled within-subject crossover study to test the effects of varenicline during smoking and abstinence. Thirty-two subjects participated in the ERP study, with half receiving placebo first and half varenicline first (VP). RESULTS: Nicotine and varenicline enhanced mouse P20 amplitude, while nicotine improved P20 habituation by selectively increasing the first-click response. Similar to mice, abstinence reduced P50 habituation relative to smoking by reducing the first-click response. There was no effect of varenicline on P50 amplitude during abstinence across subjects. However, there was a significant effect of medication order on P50 amplitude during abstinence. Subjects in the PV group displayed reduced P50 during abstinence, which was blocked by varenicline. However, subjects in the VP group did not display abstinence-induced P50 reduction. CONCLUSIONS: Data suggest that smoking improves sensory processing. Varenicline mimics amplitude changes associated with nicotine and smoking but fails to alter habituation. The effect of medication order suggests a possible carryover effect from the previous arm. This study supports the predictive validity of ERPs in mice as a marker of drug effects in human studies.

Neuregulin 1 transgenic mice display reduced mismatch negativity, contextual fear conditioning and social interactions.

INTRODUCTION: Neuregulin-1 (NRG1) is one of susceptibility genes for schizophrenia and plays critical roles in glutamatergic, dopaminergic and GABAergic signaling. Using mutant mice heterozygous for Nrg1 (Nrg1(+/-)) we studied the effects of Nrg1 signaling on behavioral and electrophysiological measures relevant to schizophrenia. EXPERIMENTAL PROCEDURE: Behavior of Nrg1(+/-) mice and their wild type littermates was evaluated using pre-pulse inhibition, contextual fear conditioning, novel object recognition, locomotor, and social choice paradigms. Event-related potentials (ERPs) were recorded to assess auditory gating and novel stimulus detection. RESULTS: Gating of ERPs was unaffected in Nrg1(+/-) mice, but mismatch negativity in response to novel stimuli was attenuated. The Nrg1(+/-) mice exhibited behavioral deficits in contextual fear conditioning and social interactions, while locomotor activity, pre-pulse inhibition and novel object recognition were not impaired. SUMMARY: Nrg1(+/-) mice had impairments in a subset of behavioral and electrophysiological tasks relevant to the negative/cognitive symptom domains of schizophrenia that are thought to be influenced by glutamatergic and dopaminergic neurotransmission. These mice are a valuable tool for studying endophenotypes of schizophrenia, but highlight that single genes cannot account for the complex pathophysiology of the disorder.

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons.

Calcium ions (Ca(2+)) released from inositol trisphosphate (IP(3))-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP(3) receptor (IP(3)R) antagonists represent an important tool for dissociating these consequences of IP(3) generation and IP(3)R-dependent internal Ca(2+) release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca(2+) sources. In this study, we have described the actions of the IP(3)R and store-operated Ca(2+) channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca(2+) release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 microM) sufficient for attenuating or blocking IP(3)-mediated internal Ca(2+) release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca(2+)-dependent potassium (K(+)) conductances.

Role of beta2-containing nicotinic acetylcholine receptors in auditory event-related potentials.

RATIONALE: Nicotine improves sensory processing in schizophrenic individuals, as measured by changes in auditory event-related potentials (ERPs). Nicotine administration also alters ERPs in mice by increasing the amplitude and gating of the P20 ERP component while decreasing the amplitude of the N40 ERP component. Less is known about the role of specific nicotinic acetylcholine receptor (nAChR) subtypes. OBJECTIVES: In this study, we examined whether nAChRs containing the beta2 subunit contribute to nicotine's effects on auditory ERPs. MATERIALS AND METHODS: We tested the effect of nicotine in wild-type mice and mice lacking the beta2 nAChR subunit. Mice underwent stereotaxic implantation of stainless steel electrodes located in the CA3 region of the hippocampus, and 50 paired click stimuli were delivered during each drug condition. RESULTS: There was no significant difference in P20 or N40 amplitude or gating between genotypes during the control condition, suggesting that beta2-containing receptors are not essential for the baseline auditory ERP response. Nicotine increased P20 amplitude and enhanced gating in wild-type and beta2 knockout mice, but only decreased N40 amplitude in wild-type mice. There was no effect of nicotine on N40 gating in either genotype. CONCLUSIONS: beta2-containing receptors are necessary for nicotine's effects on the N40 component of the mouse auditory ERP. These results suggest that beta2-containing nAChRs modulate sensory processing and may serve as a therapeutic target in schizophrenic individuals.

Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria.

BACKGROUND: Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group. RESULTS: We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other alpha-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds. CONCLUSION: SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.