Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons.

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques - karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq - to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

Optimized CRISPR inhibition and activation opens key avenues for systematic biological exploration in zebrafish.

The application of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) technologies in zebrafish has the potential to expand its capacity for the study of gene function significantly. We have developed a codon optimized CRISPRi/a for zebrafish; here we provide proof-of-principle data across established pigmentary and growth phenotypes. We established a zebrafish codon-optimized cas9 gene, harboring mutations D10A and D839A to render the protein catalytically inactive ( dCas9 ). Similarly, codon-optimized Krüppel associated box (KRAB) and methylated CP2 (MeCP2) inactivating domains or VP64 activator domain were cloned downstream from dCas9 for CRISPRi and CRISPRa, respectively. To validate CRISPRi, we targeted key genes in melanocyte differentiation ( sox10, mitfa, and mitfb) ; and melanin production (tyrosinase; tyr ). Microinjection of CRISPRi mRNA and single guide RNAs (sgRNAs) targeting the tyr promoter or 5'-UTR resulted in larvae with hypopigmented epidermal melanocytes. Transcription factors mitfa and mitfb similarly direct differentiation and pigmentation of epidermal melanocytes ( mitfa ) and retinal pigment epithelium (RPE, mitfb ). CRISPRi-mediated targeting of their promoters or 5'-UTR also results in pronounced hypopigmentation of epidermal melanocytes ( mitfa ), and RPE ( mitfb ). So too, targeting CRISPRi to the sox10 promoter results in hypopigmentation of both epidermal melanocytes and RPE consistent with its role upstream of mitfa and mitfb , and tyr . Finally, we asked whether CRISPRi and CRISPRa could be used to modulate a single gene, to yield hypomorphic and hypermorphic effects, selecting mrap2a , as our target. This gene regulates energy homeostasis and somatic growth via inhibition of the melanocortin 4 receptor gene ( mc4r ). We demonstrate that targeting the mrap2a 5'-UTR with CRISPRa or CRISPRi significantly increases or decreases larval body length, respectively. We demonstrate the utility of CRISPRi/a for modulating control of zebrafish gene expression, facilitating efficient assay of candidate gene function and disease relevance.

Multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci.

Genome-wide association studies (GWASs) have identified hundreds of risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWASs, and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotypes to identify quantitative trait loci for expression (eQTLs) and splicing (sQTLs) in coronary arteries from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary artery; 19% exhibited cell-type-specific expression. Colocalization revealed subgroups of eGenes unique to CAD and blood pressure GWAS. Fine-mapping highlighted additional eGenes, including TBX20 and IL5. We also identified sQTLs for 1,690 genes, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing to accurately identify disease-relevant isoform expression. Our work provides a patient-derived coronary artery eQTL resource and exemplifies the need for diverse study populations and multifaceted approaches to characterize gene regulation in disease processes.

Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons.

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate of disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques - karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq - to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo , mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable a proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons.

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate of disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques - karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq - to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo , mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable a proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes.

Integrative multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci.

Genome-wide association studies (GWAS) have identified hundreds of genetic risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWAS and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotype information to identify quantitative trait loci (QTL) for gene expression and splicing in coronary arteries obtained from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary arteries and 19% exhibited cell-type-specific expression. Colocalization analysis with GWAS identified subgroups of eGenes unique to CAD and blood pressure. Fine-mapping highlighted additional eGenes of interest, including TBX20 and IL5 . Splicing (s)QTLs for 1,690 genes were also identified, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing events to accurately identify disease-relevant gene expression. Our work provides the first human coronary artery eQTL resource from a patient sample and exemplifies the necessity of diverse study populations and multi-omic approaches to characterize gene regulation in critical disease processes.

Enhanced single-cell RNA-seq workflow reveals coronary artery disease cellular cross-talk and candidate drug targets.

BACKGROUND AND AIMS: The atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability are not yet available. We sought to develop a scRNA-seq analysis workflow to investigate this environment and uncover potential therapeutic approaches. We designed a user-friendly, reproducible workflow that will be applicable to other disease-specific scRNA-seq datasets. METHODS: Here we incorporated automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into a ready-to-deploy workflow. We applied this pipeline to further investigate a previously published human coronary single-cell dataset by Wirka et al. Notably, we developed an interactive web application to enable further exploration and analysis of this and other cardiovascular single-cell datasets. RESULTS: We revealed distinct derivations of fibroblast-like cells from smooth muscle cells (SMCs), and showed the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several avenues for future pharmacological development for precision medicine. Further, our interactive web application, PlaqView (www.plaqview.com), allows lay scientists to explore this and other datasets and compare scRNA-seq tools without prior coding knowledge. CONCLUSIONS: This publicly available workflow and application will allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. Our analysis pipeline provides many hypothesis-generating tools to unravel the etiology of coronary artery disease. We also highlight potential mechanisms for several drugs in the atherosclerotic cellular environment. Future releases of PlaqView will feature more scRNA-seq and scATAC-seq atherosclerosis-related datasets to provide a critical resource for the field, and to promote data harmonization and biological interpretation.

Latin American consensus on guidelines for chronic migraine treatment.

Chronic migraine is a condition with significant prevalence all around the world and high socioeconomic impact, and its handling has been challenging neurologists. Developments for understanding its mechanisms and associated conditions, as well as that of new therapies, have been quick and important, a fact which has motivated the Latin American and Brazilian Headache Societies to prepare the present consensus. The treatment of chronic migraine should always be preceded by a careful diagnosis review; the detection of possible worsening factors and associated conditions; the stratification of seriousness/impossibility to treat; and monitoring establishment, with a pain diary. The present consensus deals with pharmacological and nonpharmacological forms of treatment to be used in chronic migraine.

Latin American consensus on guidelines for chronic migraine treatment / Consenso latino-americano para as diretrizes de tratamento da migranea cronica

Chronic migraine is a condition with significant prevalence all around the world and high socioeconomic impact, and its handling has been challenging neurologists. Developments for understanding its mechanisms and associated conditions, as well as that of new therapies, have been quick and important, a fact which has motivated the Latin American and Brazilian Headache Societies to prepare the present consensus. The treatment of chronic migraine should always be preceded by a careful diagnosis review; the detection of possible worsening factors and associated conditions; the stratification of seriousness/impossibility to treat; and monitoring establishment, with a pain diary. The present consensus deals with pharmacological and nonpharmacological forms of treatment to be used in chronic migraine.

A migrânea crônica é uma condição com prevalência significativa ao redor do mundo e alto impacto socioeconômico, sendo que seu manuseio tem desafiado os neurologistas. Os avanços na compreensão de seus mecanismos e das condições a ela associadas, bem como nas novas terapêuticas, têm sido rápidos e importantes, fato que motivou as Sociedades Latino-americana e Brasileira de Cefaleia a elaborarem o presente consenso. O tratamento da migrânea crônica deve ser sempre precedido por uma revisão cuidadosa do diagnóstico, pela detecção de possíveis fatores de piora e das condições associadas, pela estratificação de gravidade/impossibilidade de se tratar e pelo monitoramento com um diário da dor. Este consenso apresenta abordagens farmacológicas e não-farmacológicas para tratar a migrânea crônica.

[Familial amyloidotic polyneuropathy type I]. / Polineuropatía amiloidótica familiar tipo I.

Familial amyloidotic polyneuropathy type I is an autosomal dominant inherited disorder characterized by progressive peripheral and autonomic neuropathy, associated with neural and systemic amyloid deposits. The abnormality usually lies in the transthyretin (TTR) gene. We report a 25 years old man with 18 months history of dysesthesias and pain in the toes, abnormal micturition and sexual dysfunction. Neurophysiologically studies disclosed a sensory-motor axonal polyneuropathy. Autonomic tests showed sympathetic and parasympathetic involvement. An electron micrograph of sural nerve revealed amyloid fibrils in the endoneurium. His mother died after a clinical history suggestive, in retrospect, of familial amyloidotic polyneuropathy type I. The clinical and genetic analysis of this cause of polyneuropathy are discussed.

Seguimiento de un año en pacientes parkinsonianos tratados con electroterapia convulsiva / Follow-up of 1 year in parkinsonian patients treated with electroconvulsive therapy

Se reporta el seguimiento de un año del efecto sobre los síntomas motores en 3 pacientes portadores de una enfermedad de parkinson que fueron tratados con terapia electroconvulsiva. Los pacientes fueron evaluados uno, seis y doce meses posteriores a la terapia electroconvulsiva y los resultados sugieren el efecto benéfico sobre los síntomas motores de la enfermedad de parkinson que resulta limitado en el tiempo a menos de 6 meses. Se discute el papel actual de este tipo de terapia en el manejo de la enfermedad de parkinson

Evaluación de los efectos de la desmopresina intranasal para el manejo de casos con poliaquiuria en la enfermedad de Parkinson: presentación de tres casos clínicos / The effects of nasal desmopressin in the management of polyuria in Parkinson's disease: report of 3 clinical cases

Los pacientes con EP pueden presentar poliaquiuria y poliuria nocturna en un 38 a 71 por ciento, un síntoma que interfiere con el sueño. Material y métodos: Se evaluaron 3 pacientes con enfermedad de Parkinson idiopática, que presentaban poliaquiuria nocturna. Se descartó infección urinaria y enfermedades nefrourológicas. Dos hombres y una mujer de 67, 67 y 77 años de edad, con entre 14 y 17 años de enfermedad de Parkinson y en tratamiento con levodopa. Todos los casos eran avanzados con Hoehn y Yahr, 4 con complicaciones evolutivas de la EP. Se le prescribe desmopresina ascetato en spray nasal, 10 µg intranasal por una semana al acostarse, la segunda semana 20 µg. Resultados. Nuestros dos casos presentaron una buena respuesta sintomática con una disminución del número de veces que orinaron en la noche. Em ambos casos hubo una clara redistribución del volumen de orina de 24 horas, con una disminución del 30 por ciento del volumen nocturno. En ambos casos no hubo cambios significativos en el sodio y la osmolaridad plasmática. Caso 3. Este caso suspendió la terapia por presentar severa exacerbación de las distonías dolorosas que el paciente presentaba previamente. Si bien el número de pacientes estudiados en esta experiencia no permite conclusiones definitivas, los resultados obtenidos hacen plantear que el uso de desmopresina intranasal en algunos pacientes parkinsonianos puede ser una herramienta terapéutica útil, la cual debe ser monitorizada clínicamente y con el laboratorio, para prevenir desequilibrio hidroelectrolítico como la hiponatremia

Análisis de la estabilidad de soluciones, a diferentes concentraciones de levodopa más carbidopa / Analysis of the solutions estability to different concentrations of levodopa plus carbidopa

La levodopa continúa siendo la terapia más efectiva en la enfermedad de Parkinson (EP), el desarrollo de diferentes formulaciones ha respondido a las distintas necesidades de los pacientes con EP avanzada. Las formulaciones líquidas o dispersables se han orientado a optimizar la farmacocinética periférica de la levodopa. Se diseñaron 4 experiencias diferentes: la primera con soluciones a diferentes concentraciones de levodopa (1 a 4 mg/ml); el segundo grupo soluciones a 1 mg/ml de levodopa con y sin filtrarlas; el tercero, igual concentración con las diferentes formas comerciales; y 4 las soluciones preparadas por los pacientes

Evaluación de la utilidad de la dieta rica en fibras para el manejo de la constipación en la enfermedad de Parkinson / Useful assessment of a fiber rich diet to treat constipation in Parkinson's disease

Reportamos un estudio en que se evalúa el uso de dieta rica en fibras (DRF) en el manejo de la constipación en la enfermedad de Parkinson (EP).Se evalúan 14 pacientes con edad media 66.5ñ6.7 años, tiempo de evolución de 7.5ñ5.6. La escala unificada para evaluar EP en su punto III tiene una media de 39.9ñ9.6 puntos. Todos los pacientes presentaban contipación y se les instruyó para consumir DRF (30 gr), fueron evaluados en forma basal y posteriormente al mes. El número de evacuaciones espontáneas antes del uso de DRF fue de 0.5ñ1.0 por semana y al mes de usar DRF 2.2ñ2.0 (p<0.05). Otros parámetros como consistencia de las heces, esfuerzo defecatorio y uso de laxantes presentaron mejprías significativas. La frecuencia de presentación de dolor o disconfort en la defecación y las evacuaciones incompletas no presentaron variaciones de significación