A stochastic RNA editing process targets a limited number of sites in individual Drosophila glutamatergic motoneurons.

RNA editing is a post-transcriptional source of protein diversity and occurs across the animal kingdom. Given the complete profile of mRNA targets and their editing rate in individual cells is unclear, we analyzed single cell RNA transcriptomes from Drosophila larval tonic and phasic glutamatergic motoneuron subtypes to determine the most highly edited targets and identify cell-type specific editing. From ∼15,000 genes encoded in the genome, 316 high confidence A-to-I canonical RNA edit sites were identified, with 102 causing missense amino acid changes in proteins regulating membrane excitability, synaptic transmission, and cellular function. Some sites showed 100% editing in single neurons as observed with mRNAs encoding mammalian AMPA receptors. However, most sites were edited at lower levels and generated variable expression of edited and unedited mRNAs within individual neurons. Together, these data provide insights into how the RNA editing landscape alters protein function to modulate the properties of two well-characterized neuronal populations in Drosophila .

Molecular logic of synaptic diversity between Drosophila tonic and phasic motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features are poorly understood. To identify molecular pathways that contribute to synaptic diversity, single-neuron Patch-seq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated that synaptic active zones in phasic motoneurons are more compact and display enhanced Ca2+ influx compared with their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications, and intracellular Ca2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Confirmed Fatal Colchicine Poisoning in an Adolescent with Blood and Bile Concentrations-Implications for GI Decontamination?

INTRODUCTION: Colchicine is commonly used to treat diseases like acute gouty arthritis. However, colchicine has a very narrow therapeutic index, and ingestions of > 0.5mg/kg can be deadly. We report a fatal acute colchicine overdose in an adolescent. Blood and postmortem bile colchicine concentrations were obtained to better understand the degree of enterohepatic circulation of colchicine. CASE REPORT: A 13-year-old boy presented to the emergency department after acute colchicine poisoning. A single dose of activated charcoal was administered early but no other doses were attempted. Despite aggressive interventions such as exchange transfusion and veno-arterial extracorporeal membrane oxygenation (VA-ECMO), the patient died 8 days later. Postmortem histology was notable for centrilobular necrosis of the liver and a cardiac septal microinfarct. The patient's blood colchicine concentration on hospital days 1 (~30 hours post-ingestion), 5, and 7 was 12ng/mL, 11ng/mL, and 9.5ng/mL, respectively. A postmortem bile concentration obtained during autopsy was 27ng/mL. DISCUSSION: Humans produce approximately 600mL of bile daily. Assuming that activated charcoal would be able to adsorb 100% of biliary colchicine, using the bile concentration obtained above, only 0.0162mg of colchicine per day would be able to be adsorbed and eliminated by activated charcoal in this patient. CONCLUSION: Despite supportive care, activated charcoal, VA-ECMO, and exchange transfusion, modern medicine may not be enough to prevent death in severely poisoned colchicine patients. Although targeting enterohepatic circulation with activated charcoal to enhance elimination of colchicine sounds attractive, the patient's low postmortem bile concentration of colchicine suggests a limited role of activated charcoal in enhancing elimination of a consequential amount of colchicine.

Molecular Logic of Synaptic Diversity Between Drosophila Tonic and Phasic Motoneurons.

Although neuronal subtypes display unique synaptic organization and function, the underlying transcriptional differences that establish these features is poorly understood. To identify molecular pathways that contribute to synaptic diversity, single neuron PatchSeq RNA profiling was performed on Drosophila tonic and phasic glutamatergic motoneurons. Tonic motoneurons form weaker facilitating synapses onto single muscles, while phasic motoneurons form stronger depressing synapses onto multiple muscles. Super-resolution microscopy and in vivo imaging demonstrated synaptic active zones in phasic motoneurons are more compact and display enhanced Ca 2+ influx compared to their tonic counterparts. Genetic analysis identified unique synaptic properties that mapped onto gene expression differences for several cellular pathways, including distinct signaling ligands, post-translational modifications and intracellular Ca 2+ buffers. These findings provide insights into how unique transcriptomes drive functional and morphological differences between neuronal subtypes.

Peripheral complement interactions with amyloid ß peptide in Alzheimer's disease: Polymorphisms, structure, and function of complement receptor 1.

INTRODUCTION: Genome-wide association studies consistently show that single nucleotide polymorphisms (SNPs) in the complement receptor 1 (CR1) gene modestly but significantly alter Alzheimer's disease (AD) risk. Follow-up research has assumed that CR1 is expressed in the human brain despite a paucity of evidence for its function there. Alternatively, erythrocytes contain >80% of the body's CR1, where, in primates, it is known to bind circulating pathogens. METHODS: Multidisciplinary methods were employed. RESULTS: Conventional Western blots and quantitative polymerase chain reaction failed to detect CR1 in the human brain. Brain immunohistochemistry revealed only vascular CR1. By contrast, erythrocyte CR1 immunoreactivity was readily observed and was significantly deficient in AD, as was CR1-mediated erythrocyte capture of circulating amyloid ß peptide. CR1 SNPs associated with decreased erythrocyte CR1 increased AD risk, whereas a CR1 SNP associated with increased erythrocyte CR1 decreased AD risk. DISCUSSION: SNP effects on erythrocyte CR1 likely underlie the association of CR1 polymorphisms with AD risk.

Peripheral complement interactions with amyloid ß peptide in Alzheimer's disease: 2. Relationship to amyloid ß immunotherapy.

INTRODUCTION: Our previous studies have shown that amyloid ß peptide (Aß) is subject to complement-mediated clearance from the peripheral circulation, and that this mechanism is deficient in Alzheimer's disease. The mechanism should be enhanced by Aß antibodies that form immune complexes (ICs) with Aß, and therefore may be relevant to current Aß immunotherapy approaches. METHODS: Multidisciplinary methods were employed to demonstrate enhanced complement-mediated capture of Aß antibody immune complexes compared with Aß alone in both erythrocytes and THP1-derived macrophages. RESULTS: Aß antibodies dramatically increased complement activation and opsonization of Aß, followed by commensurately enhanced Aß capture by human erythrocytes and macrophages. These in vitro findings were consistent with enhanced peripheral clearance of intravenously administered Aß antibody immune complexes in nonhuman primates. DISCUSSION: Together with our previous results, showing significant Alzheimer's disease deficits in peripheral Aß clearance, the present findings strongly suggest that peripheral mechanisms should not be ignored as contributors to the effects of Aß immunotherapy.

Peripheral complement interactions with amyloid ß peptide: Erythrocyte clearance mechanisms.

INTRODUCTION: Although amyloid ß peptide (Aß) is cleared from the brain to cerebrospinal fluid and the peripheral circulation, mechanisms for its removal from blood remain unresolved. Primates have uniquely evolved a highly effective peripheral clearance mechanism for pathogens, immune adherence, in which erythrocyte complement receptor 1 (CR1) plays a major role. METHODS: Multidisciplinary methods were used to demonstrate immune adherence capture of Aß by erythrocytes and its deficiency in Alzheimer's disease (AD). RESULTS: Aß was shown to be subject to immune adherence at every step in the pathway. Aß dose-dependently activated serum complement. Complement-opsonized Aß was captured by erythrocytes via CR1. Erythrocytes, Aß, and hepatic Kupffer cells were colocalized in the human liver. Significant deficits in erythrocyte Aß levels were found in AD and mild cognitive impairment patients. DISCUSSION: CR1 polymorphisms elevate AD risk, and >80% of human CR1 is vested in erythrocytes to subserve immune adherence. The present results suggest that this pathway is pathophysiologically relevant in AD.

Interactions with Astroglia Influence the Shape of the Developing Dendritic Arbor and Restrict Dendrite Growth Independent of Promoting Synaptic Contacts.

Astroglia play key roles in the development of neurons, ranging from regulating neuron survival to promoting synapse formation, yet basic questions remain about whether astrocytes might be involved in forming the dendritic arbor. Here, we used cultured hippocampal neurons as a simple in vitro model that allowed dendritic growth and geometry to be analyzed quantitatively under conditions where the extent of interactions between neurons and astrocytes varied. When astroglia were proximal to neurons, dendrites and dendritic filopodia oriented toward them, but the general presence of astroglia significantly reduced overall dendrite growth. Further, dendritic arbors in partial physical contact with astroglia developed a pronounced pattern of asymmetrical growth, because the dendrites in direct contact were significantly smaller than the portion of the arbor not in contact. Notably, thrombospondin, the astroglial factor shown previously to promote synapse formation, did not inhibit dendritic growth. Thus, while astroglia promoted the formation of presynaptic contacts onto dendrites, dendritic growth was constrained locally within a developing arbor at sites where dendrites contacted astroglia. Taken together, these observations reveal influences on spatial orientation of growth as well as influences on morphogenesis of the dendritic arbor that have not been previously identified.