Queremos reconocer el espíritu de cooperación de todos los miembros Caldwell laboratorio. Movimiento de investigación trastornos en el laboratorio ha sido apoyado por la distonía Bachmann-Strauss y la Fundación Parkinson, Reino Parkinson Foundation, American Parkinson Disease Association, Asociación de la Enfermedad de Parkinson de Alabama, la Fundación Michael J. Fox para la Investigación del Parkinson, y una investigación de Pregrado

A. Construcción de plásmido de expresión Dos plásmidos son necesarios: una para la expresión específica de tejido del gen de interés y un segundo como marcador de transformación seleccionables (aunque el marcador plásmido está generalmente disponible dentro de la comunidad de investigación). Experimental plásmido <ol><li> Seleccione un promotor que se expresa en el tipo de tejidos / células de interés, en este caso, el transportador de DA (Pdat-1) se utiliza el promotor. Este plásmido de expresión fue creada como una puerta de enlace Invitrogen sistema compatible con vector destino (pDEST-DAT-1) para permitir la inserción de un gen de interés aguas abajo del promotor de la clonación de recombinación 1. </li><li> El cDNA del gen de interés es amplificado por PCR utilizando los cebadores que contienen la puerta de enlace att secuencia B de recombinación. El cDNA amplificado por primera vez se recombinan en el vector de donantes pDONR201 para crear el vector de entrada y luego se transformó en la cepa E. coli DH5a. Después de la selección de recombinantes éxito y mini-prep aislamiento de ADN, el vector de entrada es recombinada en el pDEST-DAT-1 vector de destino para crear el plásmido de expresión. Detalles sobre la clonación de recombinación están disponibles, ya sea el manual de Invitrogen Gateway o en Caldwell et al. 5. </li></ol> Marcador seleccionable plásmido Un marcador plásmido transgénico consiste en un vector con un promotor que determinan la expresión de una proteína fluorescente en un tejido obvio. En este procedimiento en particular, el promotor unc-54 unidades de expresión de la proteína de cerezo en el cuerpo de la pared muscular (punción-54:: cereza). B. Generación de transgénicos a través de C. elegans microinyección Ver artículo relacionado Jove: <a href="https://jove.unicartagenaproxy.elogim.com/index/details.stp?ID=833">https://jove.unicartagenaproxy.elogim.com/index/details.stp?ID=833</a> C. cruza genética para el análisis de la neurodegeneración DA <ol><li> Los gusanos son cultivados usando procedimientos estándar de 6. </li><li> Lugar 10 Pdat-1:: α-syn, Pdat-1:: GFP los hombres 1, 2 sobre una placa de acoplamiento con 4.3 L4 etapa hermafroditas transgénicos (que expresa Pdat-1:: gen X y punción de 54:: cherry) . Esto debe realizarse de forma individual para cada una de las tres líneas separadas estable creado. Después de dos días, eliminar a los machos. </li><li> Inspeccione la generación F1, si hay varios hijos varones, el apareamiento tuvo éxito. </li><li> Clon de 5 hermafrodita L4 animales de cada cruz que exhiben tanto el Pdat-1:: GFP fluorescentes marcador (heredado de los padres varones) y el 54 punción:: cherry pared fluorescentes cuerpo marcador de músculo (se hereda de los padres hermafrodita). Que los animales clonados tienen que estar en la etapa de L4 para asegurarse de que aún no han apareado con machos presentes en la placa. Les permitan auto-cross y producir una progenie F2. </li><li> F2 animales producidos en el paso 3 se clonan a cabo. En concreto, la transferencia de los animales ~ 5.10 que exhiben ambos marcadores fluorescentes para sus platos individuales. Pantalla de la generación F3 para las placas donde el 100% de la GFP animales expresan en las neuronas DA y algunos de los animales se expresa la cereza en las células del cuerpo músculo de la pared. Estos animales serán homocigotos para el Pdat-1:: α-syn, Pdat-1:: gen GFP, mientras que de forma estable la transmisión de la recién creada transgén (gen X). </li></ol> D. Análisis de las neuronas dopaminérgicas <ol><li> Prepare un plato fresco para cada una de las tres líneas creadas anteriormente, así como la Pdat-1:: α-syn, Pdat-1:: GFP sola cepa. Les permiten crecer a la edad adulta. </li><li> La transferencia de los adultos 30-40 transgénicos de cada línea en un plato fresco. Les permitan sentar los embriones de 4 horas a 20 º C. </li><li> Retire los adultos. Permita que los embriones para desarrollar durante 3-4 días a 20 º C hasta que alcancen la etapa de L4. </li><li> Elige ~ 100 L4 transgénicos animales a una placa que contiene 0,04 mg / ml 5-fluoro-2&#39;-desoxiuridina (FUDR). Este desarrollo nucleótidos bloques analógicos de la próxima generación a través de la inhibición de la síntesis de ADN, evitando así la descendencia de los animales de experimentación abrumadora 7. Proteger las placas FUDR de la luz, ya que es sensible a la luz. </li><li> Los animales adultos se analizaron en varios días después de la puesta de huevos. El día de análisis apropiados se determinan empíricamente. Por ejemplo, hemos determinado que el 77%, 87% y 90% de los Pdat-1:: α-syn, Pdat-1:: GFP animales exhiben degenerado neuronas DA en los días 6, 7 y 10 de desarrollo posterior, respectivamente. Por lo tanto, si podemos predecir que el transgen de interés podría aumentar la neurodegeneración, vamos a analizar en el día 6 y / o 7. Del mismo modo, los transgenes que podrían suprimir la neurodegeneración se analizarán en los días 7 y 10. </li><li> Hacer 4 pastillas de dulce de agarosa (a diferencia de las pastillas de microinyección agar, estos se utilizan frescos y no permitir que se seque). Se establecen dos portaobjetos de microscopio que tienen un pedazo de cinta a través de ellas, la cinta sirve como separador de pastillas de equivalente de espesor. Ponga un terciodeslice entre ellos en el banco (que se colocan de tres al día). Coloque una gota de agarosa fundida en el centro de la diapositiva y rápidamente poner otra diapositiva en la parte superior de la agarosa, perpendicular y en las tres diapositivas. Hacer varias almohadillas adicionales, dejando a los dos portaobjetos, hasta que la pastilla se encuentra listo para su uso. </li><li> Para el análisis de gusano neuronas DA, el lugar una caída de 6 l de levamisol mm 3 (un anestésico) en un 22 x 30 mm cubierta de vidrio. Recoger 40 animales adultos (10 extra más allá de los 30 que anotó) dentro de la gota, y luego invertir la cubierta de cristal en la almohadilla de agarosa. Repita este procedimiento para cada una de las otras líneas. </li><li> Puntuación 30 animales por cada línea de la presencia de cada CEP y la neurona ADE utilizando un microscopio compuesto con epifluorescencia y un cubo de filtro que permite la visualización del FITC o GFP. Nosotros usamos un filtro de ganar a las buenas prácticas agrarias HyQ cubo (Chroma Technology). Anota los datos en la hoja de registro adjunta. Animales de tipo salvaje mantendrá fluorescencia de GFP completa en todos los CEP 4 y 2 neuronas ADE. Animales individuales mostrando la pérdida de neuronas DA se califican como no-WT o &quot;degeneración&quot;. Del mismo modo, el grado de degeneración se puede marcar, contando el total de las neuronas perdidas en cada animal, así como a la población. </li><li> Repetir el análisis dos veces más (30 animales más por línea de prueba). El número total de gusanos de tipo salvaje exhibiendo las neuronas DA de las tres rondas de análisis es el promedio. El análisis estadístico para la neuroprotección continuación, se realiza utilizando la t de Student-test (p &lt;0,05) para comparar los gusanos de control (Pdat-1:: α-syn, Pdat-1:: GFP) con las cepas que sobreexpresan genes en las neuronas DA . </li></ol>

<ol>
	<li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans.">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li><li>Hamamichi, S., Rivas, R. N., Knight, A. L., Cao, S., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis-based+RNAi+screening+identifies+neuroprotective+genes+in+a+Parkinson&#x2019;s+disease+model.">Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson&#x2019;s disease model.</a> PNAS. 105, 728-733 (2008).</li><li>Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-synuclein+blocks+ER-golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. -. C., McCaffery, J. M., Barlowe, C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis.">The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.</a> PNAS. 105, 145-150 (2008).</li><li>Caldwell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Genomics:+A+Discovery-Based+Laboratory+Course.">Integrated Genomics: A Discovery-Based Laboratory Course.</a> , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Gandhi, S., Santelli, J., Mitchell, J. D. H., Stiles, J. W., Sanadi, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+maintaining+large,+aging+populations+of+Caenorhabditis+elegans.">A simple method for maintaining large, aging populations of Caenorhabditis elegans.</a> Mechanisms of Ageing and Development. 12, 137-150 (1980).</li></ol>

La pérdida depende de la edad de las neuronas de dopamina es una característica clínica de la enfermedad de Parkinson y se ha asociado con la acumulación o el mal plegamiento de una proteína llamada alfa-sinucleína. Aquí se demuestra la forma de etiqueta, a través de un transgén fluorescentes, las neuronas dopaminérgicas de la C. elegans e imitar la neurodegeneración en la enfermedad de Parkinson por coexpressing humana alfa-sinucleína en estas células. Este video muestra la metodología para el crecimiento, cr...

<table><tbody><tr><td>Agarose</td><td>Ultrapure</td><td>Invitrogen</td><td>15510-027</td><td></td></tr><tr><td>Coverglass 20x30 mm</td><td></td><td>Fisher Scientific</td><td>12-548-5A</td><td></td></tr><tr><td>Microscope Slides</td><td>Plain, 3x1&quot;</td><td>Fisher Scientific</td><td>12-549</td><td></td></tr><tr><td>Dissecting with Fluorescence</td><td>Microscope</td><td>Nikon Instruments</td><td>SMZ800</td><td></td></tr><tr><td>Dissecting</td><td>Microscope</td><td>Nikon Instruments</td><td>SMZ645</td><td></td></tr><tr><td>Epifluorescent</td><td>Microscope</td><td>Nikon Instruments</td><td>Model E-800</td><td></td></tr><tr><td>Filter Cube, GFP HYQ</td><td>Endow Bandpass</td><td>Chroma Technology Corp.</td><td></td><td></td></tr></tbody></table>

application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

Mejoras en el diagnóstico y el tratamiento de la enfermedad de Parkinson (EP) dependen de los conocimientos sobre factores de vulnerabilidad que hacen que las poblaciones en riesgo. En el proceso de tratar de identificar nuevos factores genéticos asociados con la enfermedad, los científicos han generado muchas listas de genes candidatos, los polimorfismos y proteínas que representan importantes avances, pero estos conductores siguen mecánicamente indefinido. Nuestro trabajo está dirigido a reducir significativamente estas listas mediante la explotación de las ventajas de un sistema modelo animal simple. Mientras que los humanos tienen miles de millones de neuronas, la lombriz Caenorhabditis elegans microscópica tiene precisamente 302, de los cuales sólo ocho producir dopamina (DA) en hemaphrodites. Expresión de un gen humano que codifica la proteína asociada a la EP, la alfa-sinucleína, en C. elegans resultados neuronas DA de la dosis y la neurodegeneración dependiente de la edad. Gusanos expresar humana alfa-sinucleína en las neuronas DA son isogénicas y expresar tanto las buenas prácticas agrarias y los derechos humanos alfa-sinucleína en el promotor del transportador de DA (Pdat-1). La presencia de GFP sirve como un marcador visualiza fácilmente para el seguimiento de la neurodegeneración DA en estos animales. Inicialmente se demostró que la alfa-sinucleína la neurodegeneración inducida por DA podrían ser rescatados en estos animales por torsinA, una proteína con actividad chaperona molecular 1. Además, el candidato PD relacionadas con los genes identificados en nuestro laboratorio a través de grandes esfuerzos de detección RNAi mediante una prueba de mal plegamiento de la alfa-sinucleína eran entonces sobre-expresado en C. elegans neuronas DA. Se determinó que cinco de los siete genes probados representado moduladores candidato importante de la EP, ya que rescató a la alfa-sinucleína inducida por la neurodegeneración DA 2. Además, el laboratorio de Lindquist (este número de Jove) ha llevado a cabo las pantallas de la levadura por el que la alfa-sinucleína toxicidad dependiente se utiliza como una lectura de los genes que pueden aumentar o suprimir la citotoxicidad. Nos examinó a continuación los genes candidatos de la levadura en nuestro ensayo de C. elegans neurodegeneración alfa-sinucleína y la inducida por validado con éxito muchos de estos tres objetivos, 4. Nuestra metodología se basa en la generación de un vector de expresión de C. elegans DA neuronal específica con la clonación de cDNAs de recombinación de genes candidatos bajo el control del Pdat-1 promotor. Estos plásmidos son entonces microinyección de tipo salvaje (N2) gusanos, junto con un marcador de selección para la transformación exitosa. Múltiples líneas transgénicas estables de producir la proteína en las neuronas DA candidato se obtienen y se cruzó de manera independiente en la cepa de la alfa-sinucleína degenerativas y evaluados por la neurodegeneración, en el animal y el nivel de las neuronas individuales, en el curso del envejecimiento.

Watch this Scientific Journal Video about Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes at JoVE.com

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Este video muestra cómo utilizar C. elegans para evaluar la neurodegeneración nerviosas dopaminérgicas como un modelo para la enfermedad de Parkinson. Además, las pantallas de genética se utilizan para definir los factores que mejoran la degeneración o neuroprotectoras.

Aplicación de un ensayo de C. elegans la degeneración neuronal de dopamina para la validación del potencial de la enfermedad de Parkinson genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

application-c-elegans-dopamine-neuron-degeneration-assay-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

16.0K vistas.  University of Alabama. Este video muestra cómo utilizar C. elegans para evaluar la neurodegeneración nerviosas dopaminérgicas como un modelo para la enfermedad de Parkinson. Además, las pantallas de genética se utilizan para definir los factores que mejoran la degeneración o neuroprotectoras.

Video: Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

Evaluación de la función sensoriomotora en modelos de ratón de la enfermedad de Parkinson

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

Investigación

Comportamiento

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

Natación parálisis inducida a evaluar dopamina señalización en Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Modelos De Interacción Gen-Ambiente Para Desenmascarar Los Mecanismos De Susceptibilidad En La Enfermedad De Parkinson

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Medicina

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

La evaluación de fenotipos neurodegenerativas en<em&gt; Drosophila</em&gt; Las neuronas dopaminérgicas mediante ensayos de Escalada y inmunotinción de todo el cerebro

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Neurociencias

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Cultivo primario de ratón neuronas dopaminérgicas

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Análisis cuantitativo de los Defectos de escalada en un modelo de Drosophila de enfermedades neurodegenerativas

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

La identificación fiable de las neuronas dopaminérgicas de estar en el mesencéfalo culturas utilizando ARN-Secuenciación y promotor impulsado TH expresión de EGFP

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Aplicación del sistema RatWalker para el análisis de la marcha en un modelo genético de ratas de la enfermedad de Parkinson

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Cuantificación de los niveles de alteración morfológica y degeneración de la neurona dopaminérgica en Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Cultivo de neuronas dopaminérgicas del mesencéfalo humano para el perfil fenotípico

Phenotypic Profiling of Human Stem Cell-Derived Midbrain Dopaminergic Neurons

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro PD cellular models lack complexity and do not take multiple phenotypes into account. Phenotypic profiling in human induced pluripotent stem cell (iPSC)-derived mDA neurons can address these shortcomings by simultaneously measuring a range of neuronal phenotypes in a PD-relevant cell type in parallel. Here, we describe a protocol to obtain and analyze phenotypic profiles from commercially available human mDA neurons. A neuron-specific fluorescent staining panel is used to visualize the nuclear, &#945;-synuclein, Tyrosine hydroxylase (TH), and Microtubule-associated protein 2 (MAP2) related phenotypes. The described phenotypic profiling protocol is scalable as it uses 384-well plates, automatic liquid handling and high-throughput microscopy. The utility of the protocol is exemplified using healthy donor mDA neurons and mDA neurons carrying the PD-linked G2019S mutation in the Leucine-rich repeat kinase 2 (LRRK2) gene. Both cell lines were treated with the LRRK2 kinase inhibitor PFE-360 and phenotypic changes were measured. Additionally, we demonstrate how multidimensional phenotypic profiles can be analyzed using clustering or machine learning-driven supervised classification methods. The described protocol will particularly interest researchers working on neuronal disease modeling or studying chemical compound effects in human neurons.

Autor destacado: Generación de perfiles fenotípicos neuronales: un protocolo para cultivar e imagen de neuronas dopaminérgicas del mesencéfalo humano 

This protocol describes the cell culturing of human midbrain dopaminergic neurons, followed by immunological staining and the generation of neuronal phenotypic profiles from acquired microscopic high-content images allowing the identification of phenotypic variations due to genetic or chemical modulations.

Perfil fenotípico de neuronas dopaminérgicas del mesencéfalo derivadas de células madre humanas

Parkinson&#39;s disease (PD) is linked to a range of cell biological processes that cause midbrain dopaminergic (mDA) neuron loss. Many current in vitro ...