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In this Article, the top label in Fig. 5d should read 'DISH 3/16' instead of 'DISH 3/17'. This error has been corrected online.
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The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer's disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant 'genomic cDNAs' (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal 'retro-insertion' of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer's disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer's disease that were absent from healthy neurons. Neuronal gene recombination may allow 'recording' of neural activity for selective 'playback' of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Variación Genética/genética , Neuronas/citología , Neuronas/patología , Recombinación Genética , Empalme Alternativo/genética , Animales , ADN Complementario/análisis , ADN Complementario/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Especificidad de Órganos , Mutación Puntual/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia/genéticaRESUMEN
Hereditary angioedema is characterized by recurrent and unpredictable episodes of subcutaneous and mucosal swelling that can be life threatening. IONIS-PKK-LRx is a ligand-conjugated antisense oligonucleotide designed for receptor-mediated delivery to hepatocytes. In a compassionate-use pilot study, two patients with severe bradykinin-mediated angioedema were initially administered weekly subcutaneous injections of the unconjugated parent drug, IONIS-PKKRx, for 12 to 16 weeks, after which they received IONIS-PKK-LRx at a dose of 80 mg every 3 to 4 weeks for 7 to 8 months. Treatment was accompanied by a reduction in the angioedema attack rate. (Funded by Amsterdam UMC.).
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Angioedemas Hereditarios/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Precalicreína/antagonistas & inhibidores , Adulto , Angioedemas Hereditarios/metabolismo , Bradiquinina/metabolismo , Ensayos de Uso Compasivo , Femenino , Humanos , Inyecciones Subcutáneas , Oligonucleótidos Antisentido/administración & dosificación , Proyectos Piloto , Precalicreína/metabolismoRESUMEN
A new form of somatic gene recombination (SGR) has been identified in the human brain that affects the Alzheimer's disease gene, amyloid precursor protein (APP). SGR occurs when a gene sequence is cut and recombined within a single cell's genomic DNA, generally independent of DNA replication and the cell cycle. The newly identified brain SGR produces genomic complementary DNAs (gencDNAs) lacking introns, which integrate into locations distinct from germline loci. This brief review will present an overview of likely related recombination mechanisms and genomic cDNA-like sequences that implicate evolutionary origins for brain SGR. Similarities and differences exist between brain SGR and VDJ recombination in the immune system, the first identified SGR form that now has a well-defined enzymatic machinery. Both require gene transcription, but brain SGR uses an RNA intermediate and reverse transcriptase (RT) activity, which are characteristics shared with endogenous retrotransposons. The identified gencDNAs have similarities to other cDNA-like sequences existing throughout phylogeny, including intron-less genes and inactive germline processed pseudogenes, with likely overlapping biosynthetic processes. gencDNAs arise somatically in an individual to produce multiple copies; can be functional; appear most frequently within postmitotic cells; have diverse sequences; change with age; and can change with disease state. Normally occurring brain SGR may represent a mechanism for gene optimization and long-term cellular memory, whereas its dysregulation could underlie multiple brain disorders and, potentially, other diseases like cancer. The involvement of RT activity implicates already Food and Drug Administration-approved RT inhibitors as possible near-term interventions for managing SGR-associated diseases and suggest next-generation therapeutics targeting SGR elements.
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Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Encéfalo/metabolismo , Elementos de Nucleótido Esparcido Largo , ADN Polimerasa Dirigida por ARN , Recombinación Genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
The transcriptional state of a cell reflects a variety of biological factors, from cell-type-specific features to transient processes such as the cell cycle, all of which may be of interest. However, identifying such aspects from noisy single-cell RNA-seq data remains challenging. We developed pathway and gene set overdispersion analysis (PAGODA) to resolve multiple, potentially overlapping aspects of transcriptional heterogeneity by testing gene sets for coordinated variability among measured cells.
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Perfilación de la Expresión Génica/métodos , Proteoma/metabolismo , Análisis de Secuencia de ARN/métodos , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Transcriptoma/fisiología , Animales , Células Cultivadas , Simulación por Computador , Ratones , Modelos Biológicos , Modelos Estadísticos , Neuronas/fisiología , Proteoma/químicaRESUMEN
Brain somatic gene recombination (SGR) and the endogenous reverse transcriptases (RTs) that produce it have been implicated in the etiology of Alzheimer's disease (AD), suggesting RT inhibitors as novel prophylactics or therapeutics. This retrospective, proof-of-concept study evaluated the incidence of AD in people with human immunodeficiency virus (HIV) with or without exposure to nucleoside RT inhibitors (NRTIs) using de-identified medical claims data. Eligible participants were aged ≥60 years, without pre-existing AD diagnoses, and pursued medical services in the United States from October 2015 to September 2016. Cohorts 1 (N = 46,218) and 2 (N = 32,923) had HIV. Cohort 1 had prescription claims for at least one NRTI within the exposure period; Cohort 2 did not. Cohort 3 (N = 150,819) had medical claims for the common cold without evidence of HIV or antiretroviral therapy. The cumulative incidence of new AD cases over the ensuing 2.75-year observation period was lowest in patients with NRTI exposure and highest in controls. Age- and sex-adjusted hazard ratios showed a significantly decreased risk for AD in Cohort 1 compared with Cohorts 2 (HR 0.88, p < 0.05) and 3 (HR 0.84, p < 0.05). Sub-grouping identified a decreased AD risk in patients with NRTI exposure but without protease inhibitor (PI) exposure. Prospective clinical trials and the development of next-generation agents targeting brain RTs are warranted.
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Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA(A) and metabotropic GABA(B) receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity-dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA(A) and GABA(B) receptors during early development of Xenopus laevis. Using in situ hybridization and qRT-PCR, GABA(A) α2 was detected as a maternal mRNA. All other α-subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner.
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Receptores de GABA-A/genética , Receptores de GABA-B/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Fase de Segmentación del Huevo/metabolismo , Clonación Molecular , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Subunidades de Proteína/análisis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/análisis , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiología , Receptores de GABA-B/análisis , Receptores de GABA-B/metabolismo , Receptores de GABA-B/fisiología , Análisis de Secuencia de ADN , Factores de TiempoRESUMEN
The recent identification of somatic gene recombination(SGR) in human neurons affecting the well-known Alzheimer's disease (AD) pathogenic gene, amyloid precursor protein (APP), has implications for the normal and the diseased human brain. The amyloid hypothesis has been the prevailing theory for sporadic AD (SAD) pathogenesis since the discovery of APP gene involvement in familial AD and Down syndrome. Yet, despite enormous scientific and clinical effort, no disease-modifying therapy has emerged. SGR offers a novel mechanism to explain AD pathogenesis and the failures of amyloid-related clinical trials, while maintaining consistency with most aspects of the amyloid hypothesis and additionally supporting possible roles for tau, oxidative stress, inflammation, infection, and prions. SGR retro-inserts novel "genomic complementary DNAs" (gencDNAs) into neuronal genomes and becomes dysregulated in SAD, producing numerous mosaic APP variants, including DNA mutations observed in familial AD. Notably, SGR requires gene transcription, DNA strand-breaks, and reverse transcriptase (RT) activity, all of which may be promoted by well-known AD risk factors and provide a framework for the pursuit of new SGR-based therapeutics. In this perspective, we review evidence for APP SGR in AD pathogenesis and discuss its possible relevance to other AD-related dementias. Further, SGR's requirement for RT activity and the relative absence of AD in aged HIV -infected patients exposed to RT inhibitors suggest that these Food and Drug Administration (FDA)-approved drugs may represent a near-term disease-modifying therapy for AD.
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Kallikrein is the key contact system mediator responsible for the conversion of high-molecular-weight kininogen into the inflammatory vasodilator peptide bradykinin, a process regulated by C1-esterase inhibitor (C1-INH). In hereditary angioedema (HAE), genetic mutations result in deficient or dysfunctional C1-INH and dysregulation of the contact system leading to recurrent, sometimes fatal, angioedema attacks. IONIS-PKKRx is a second-generation 2'-O-(2-methoxyethyl)-modified chimeric antisense oligonucleotide, designed to bind and selectively reduce prekallikrein (PKK) mRNA in the liver. IONIS-PKKRx demonstrated dose-dependent reduction of human prekallikrein hepatic mRNA and plasma protein in transgenic mice and dose- and time-dependent reductions of plasma PKK in Cynomolgus monkeys. Similar dose-dependent reductions of plasma PKK levels were observed in healthy human volunteers accompanied by decreases in bradykinin generation capacity with an acceptable safety and tolerability profile. These results highlight a novel and specific approach to target PKK for the treatment of HAE and other diseases involving contact system activation and overproduction of bradykinin.
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Angioedemas Hereditarios/terapia , Bradiquinina/genética , Complemento C1s/genética , Precalicreína/genética , Angioedemas Hereditarios/sangre , Angioedemas Hereditarios/genética , Animales , Animales Modificados Genéticamente/sangre , Bradiquinina/sangre , Proteína Inhibidora del Complemento C1/farmacología , Complemento C1s/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Voluntarios Sanos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca fascicularis/sangre , Ratones , Oligodesoxirribonucleótidos Antisentido/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Precalicreína/antagonistas & inhibidoresRESUMEN
Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.
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Encéfalo/metabolismo , Epigénesis Genética/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Adulto , Cerebelo/metabolismo , Cerebelo/patología , Lóbulo Frontal/metabolismo , Lóbulo Frontal/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ARN , Corteza Visual/metabolismo , Corteza Visual/patologíaRESUMEN
The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain.
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Transcriptoma , Núcleo Celular , Corteza Cerebral , Perfilación de la Expresión Génica , Humanos , Neuronas , Análisis de Secuencia de ARNRESUMEN
Previous reports have shown that individual neurons of the brain can display somatic genomic mosaicism of unknown function. In this study, we report altered genomic mosaicism in single, sporadic Alzheimer's disease (AD) neurons characterized by increases in DNA content and amyloid precursor protein (APP) gene copy number. AD cortical nuclei displayed large variability with average DNA content increases of ~8% over non-diseased controls that were unrelated to trisomy 21. Two independent single-cell copy number analyses identified amplifications at the APP locus. The use of single-cell qPCR identified up to 12 copies of APP in sampled neurons. Peptide nucleic acid (PNA) probes targeting APP, combined with super-resolution microscopy detected primarily single fluorescent signals of variable intensity that paralleled single-cell qPCR analyses. These data identify somatic genomic changes in single neurons, affecting known and unknown loci, which are increased in sporadic AD, and further indicate functionality for genomic mosaicism in the CNS.