Biz tüm Caldwell Lab üyeleri işbirliği ruhu kabul etmek istiyoruz. Bachmann-Strauss Distoni ve Parkinson Vakfı, Birleşik Parkinson Vakfı, Amerikan Parkinson Hastalığı Derneği, Parkinson Hastalığı Derneği Alabama, Michael J. Fox Parkinson Araştırma Vakfı ve Lisans Araştırma laboratuarında Hareket bozuklukları araştırma tarafından desteklenmektedir

A. İfade Plazmid İnşaat İki plazmid gerekli doku özel ilgi gen ekspresyonu ve seçilebilir dönüşüm belirteci olarak ikinci bir (plazmid marker araştırma toplum içinde genellikle mevcut olmasına rağmen). Deneysel Plazmid <ol><li> Ilgi doku / hücre tipi olarak ifade edilir bir yararlanıcı seçin; DA taşıyıcı (Pdat 1) Bu durumda, organizatörü kullanılır. Bu ifade plazmid recombinational klonlama 1 organizatörü ilgi downstream herhangi bir gen ekleme sağlamak için bir Invitrogen Gateway sisteminin uyumlu hedef vektör (pDEST-DAT-1) olarak yaratılmıştır. </li><li> Ilgi genin cDNA Gateway att B recombinational dizisi içeren primerler kullanılarak amplifiye. Amplifiye cDNA ilk giriş vektörü oluşturmak için donör vektör pDONR201 içine recombined ve sonra E. coli suşu DH5α dönüştü. Başarılı rekombinantlar ve DNA mini hazırlık izolasyon seçimi takiben, giriş vektörü ifade plazmid yaratmak için pDEST-DAT-1 hedef vektör recombined. Recombinational klonlama Ayrıntılar Invitrogen Ağ Geçidi manuel veya Caldwell ve ark mevcuttur. 5. </li></ol> Plazmid Seçilebilir Marker Plazmid bir transgenik işaretleyici bariz bir doku floresan protein ifadesi itici bir yararlanıcı bir vektör oluşur. Bu özel prosedür, unc-54 organizatörü sürücüler vücut duvarı kas kiraz protein ekspresyonu (: kiraz Nokt-54). Mikroenjeksiyon yoluyla Transgenik C. elegans B. Üretimi Bkz: vallahi ilgili makale <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833</a> C. Genetik DA nörodejenerasyon Analizi için haçlar <ol><li> Worms standart prosedürler kullanarak 6 yetiştirilmektedir. </li><li> 10 Pdat-1: α-syn; Pdat-1: 3-4 aşama L4 transgenik çift cinsiyetli bir çiftleşme tabağa GFP erkeklerde 1, 2 (: gen X ve Nokt-54: Pdat-1 ifade: kiraz) . Bu oluşturulan üç ayrı istikrarlı hatları her biri için ayrı ayrı yapılmalıdır. İki gün sonra, erkek çıkarın. </li><li> F1 nesil kontrol edin; birkaç erkek döl varsa, çiftleşme başarılı oldu. </li><li> Her çapraz 5 hermafrodit L4 hayvanlar dışarı Clone sergi hem Pdat-1: GFP floresan işaretleyici (erkek ebeveyninizden miras) ve Nokt-54: kiraz floresan vücut duvarı kas işaretleyici (hermafrodit ebeveyn miras). Klonlanmış hayvanların henüz plakası üzerinde bulunan erkek hayvanlar ile evlendirilen olduğunu sağlamak için L4 aşamasında olması gerekir. Onları kendi kendine çapraz ve F2 döl üretmek izin ver. </li><li> 3. adımda üretilen F2 hayvanların klonlanmış. Özellikle, transfer ~ kendi bireysel plakaları hem flüoresan işaretleyiciler sergi 5-10 hayvanlar. Ekran hayvanların ifade DA nöronlar GFP ve bazı hayvanların% 100 vücut duvarı kas hücrelerinde ifade kiraz plakaları için F3 nesil. Bu hayvanlar Pdat-1 için homozigot olacak: α-syn; Pdat-1:: GFP geninin stably yeni oluşturulan transgen (gen X) iletimi sırasında. </li></ol> D. dopaminerjik Nöron Analizi <ol><li> Her üç satır yukarıda oluşturduğumuz yeni bir plaka, yanı sıra Pdat-1 hazırlayın: α-syn; Pdat-1: GFP yalnız gerginlik. Yetişkinliğe büyümesine izin verin. </li><li> Her satırı 30-40 transgenik yetişkin taze bir tabağa aktarın. , 20 º C&#39;de 4 saat embriyolar koymak için izin ver </li><li> Yetişkinler çıkarın. L4 aşamaya ulaşmak kadar embriyoların 20 º C&#39;de 3-4 gün geliştirmeye izin verir. </li><li> 0.04 mg / ml 5-floro-2&#39;-dezoksiuridin (FUDR) içeren bir plaka ~ 100 transgenik L4 hayvanlar seçin. DNA sentezinin inhibisyonu yoluyla yeni nesil bu nükleotid analog blok gelişimi, böylece deney hayvanları 7 ezici bir döl önler. Işığa duyarlı olduğundan ışık FUDR plakaları koruyun. </li><li> Yetişkin hayvanların yumurtlama sonrasında çeşitli gün analiz edilecektir. Ampirik olarak analiz uygun gün belirlenir. Örneğin, biz tespit ettik% 77,% 87 ve% 90 Pdat-1: α-syn Pdat-1: GFP hayvanlar sırasıyla, dejenere DA nöronları gün 6, 7 ve 10 yazılan gelişimi sergilerler. Bu nedenle, biz ilgi transgen nörodejenerasyon geliştirmek olabileceğini tahmin, biz günde 6 ve 7 / veya analiz eder. Aynı şekilde, 7 ve 10 gün nörodejenerasyon bastırmak olabilir transgenlerin analiz edilecektir. </li><li> 4 taze agaroz pedleri (mikroenjeksiyon agar pedleri aksine, bu taze kullanılan ve kurumasına izin verilmez) olun. Çapında bir parça bant iki mikroskop lamı ayarlayın; bant eşdeğer kalın pedler için ayırıcı olarak hizmet vermektedir. Üçüncü Lay(üç ayak yerleştirilmiş) bankta aralarında kaydırın. Merkezi slayt erimiş agaroz bir damla koyun ve hızlı bir şekilde dik ve her üç slayt boyunca, agaroz üstüne başka bir slayt yatıyordu. Ped kullanmak için hazır olana kadar birlikte iki slayt bırakarak, birkaç ek pedleri olun. </li><li> Solucan DA nöronları analiz etmek için, 22 x 30 mm cam kapak 3 mM levamizol (anestezi) 6 ul damla yerleştirin. Agaroz ped üzerine kapak cam invert sonra, açılan (10 30 dışında ekstra atılır) 40 yetişkin hayvanların seçin. Diğer çizgilerin her biri için yineleyin. </li><li> Puan Epifloresans ve FITC veya GFP görselleştirme sağlayan bir filtre küp ile bir bileşik mikroskop kullanılarak her CEP ve ADE nöron varlığı için hat başına 30 hayvan. GFP HYQ filtre küp (Chroma Teknolojisi) bağışlamak kullanın. Ekli puanlama kağıda kayıt verileri. Wild-tip hayvanlar tam 4 CEP GFP floresan ve 2 ADE nöronlar koruyacaktır. DA nöronların kaybı sergileyen bireysel hayvanlar olmayan WT veya &quot;dejenere&quot; olarak attı. Aynı şekilde, dejenerasyon ölçüde her hayvanın yanı sıra nüfus olarak kaybedilen toplam nöron sayarak atılır. </li><li> Analizi (deneme satır başına başına 30 daha fazla hayvan) 2 kez daha tekrarlayın. Analizler üç tur solucanlar, yabani tip DA nöronların sergileyerek toplam sayısı ortalaması alınır. Analitik nöro için istatiksel analizi daha sonra kontrolü solucanlar (: α-syn; Pdat-1: Pdat-1: GFP) karşılaştırmak için öğrenci en t-testi (p &lt;0.05) kullanılarak gerçekleştirilir. Suşları ile DA nöronların aşırı-ifade aday genler . </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>

Dopamin nöronlarının yaşa bağlı kayıp, Parkinson hastalığının bir klinik özelliğidir ve alfa-sinüklein adı verilen bir protein birikimi veya misfolding ile ilişkili bulunmuştur. Burada C dopaminerjik nöronlar, floresan transgen üzerinden, etiket nasıl göstermek elegans ve bu hücrelerin coexpressing insan alfa-sinüklein Parkinson hastalığında görülen nörodejenerasyon taklit. Bu video, büyüme, genetik geçişi, montaj ve nörodejenerasyon ya da geliştirmek veya yaşlanma boyunca nöro-...

<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

Parkinson hastalığı (PH) tanı ve tedavi gelişmeler, risk altındaki populasyonlarda hale yatkınlık faktörleri hakkında bilgi bağımlı. PD ile ilişkili yeni bir genetik faktörleri belirlemek için çalışırken sürecinde, bilim adamları, aday genler, polimorfizmleri ve önemli ilerlemeler temsil eden proteinler birçok listeler oluşturulur, ancak bu yol açar mekanistik tanımsız kalır. Bizim işimiz böyle listelere basit bir hayvan modeli sisteminin avantajlarını istismar ederek önemli ölçüde daralması doğru hedeflenmektedir. Insanlar milyarlarca nöron olmasına rağmen, mikroskopik tür solucanın Caenorhabditis elegans tam 302 var ve bunların sadece sekiz hemaphrodites üretmek dopamin (DA). İfade bir insan gen dozaj ve yaşa bağımlı nörodejenerasyon C. elegans DA nöronlar sonuçları PD-ilişkili protein, alfa-sinüklein kodlama. DA nöronların insan alfa-sinüklein ifade Worms isogenic ve DA taşıyıcı organizatörü (Pdat 1) altında GFP ve insan alfa-sinüklein ifade. GFP varlığı kolayca görüntülenebilmekte, bu hayvanlar DA nörodejenerasyon için bir marker olarak hizmet vermektedir. Biz başlangıçta alfa-sinüklein indüklenen DA nörodejenerasyon torsinA, moleküler şaperon aktivite 1 ile bir protein tarafından bu hayvanlar kurtarıldı olabileceğini gösterdi. Ayrıca, aday PD-ilişkili genlerin sonra C. elegans DA nöronların aşırı ifade edildi alfa-sinüklein misfolding assay kullanarak büyük ölçekli RNAi tarama çabaları ile laboratuvar belirlemiştir. Biz alfa-sinüklein indüklenen DA nörodejenerasyon 2 kurtardı beş yedi genlerin PD temsil önemli aday modülatörleri test olduğunu belirledi. Ayrıca, alfa-sinüklein bağımlı toksisite sitotoksisite geliştirmek veya bastırmak genler için bir okuma olarak kullanılmasını sağlayan Lindquist Lab (vallahi bu konuda) maya ekranlarında sahne aldı. Biz daha sonra C. elegans alfa-sinüklein indüklenen nörodejenerasyon tahlil maya aday genler incelenecek ve başarılı bir şekilde bu hedeflere 3, 4 çok valide . Metodoloji Pdat-1 promotör kontrolü altında aday gen cDNAs recombinational klonlama kullanarak C. elegans DA nöron spesifik ifade vektör nesil içerir. Bu plazmid sonra başarılı bir dönüşüm için seçilebilir bir işaret ile birlikte, wild-tip (N2) solucanlar microinjected. DA nöronları aday proteini üreten Çoklu istikrarlı transgenik çizgiler yaşlanma boyunca, hayvan ve bireysel nöron düzeyinde hem de elde edilen ve daha sonra bağımsız olarak geçti ve alfa-sinüklein dejeneratif gerginlik içine nörodejenerasyon açısından değerlendirildi.

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

Bu video C. elegans dopaminerjik nöron nörodejenerasyon, Parkinson hastalığı için bir model olarak değerlendirmek için nasıl kullanılacağını göstermektedir. Ayrıca, genetik ekranlar ya dejenerasyon geliştirmek veya nöroprotektif faktörleri tanımlamak için kullanılır.

Potansiyel Parkinson Hastalığı Genler Doğrulama için C. elegans Dopamin Nöron Dejenerasyonu Testi Uygulama

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

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16.0K Görüntüleme.  University of Alabama. Bu video C. elegans dopaminerjik nöron nörodejenerasyon, Parkinson hastalığı için bir model olarak değerlendirmek için nasıl kullanılacağını göstermektedir. Ayrıca, genetik ekranlar ya dejenerasyon geliştirmek veya nöroprotektif faktörleri tanımlamak için kullanılır.

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.

Parkinson Hastalığı Fare Modeller Duyusal Motor Fonksiyon Değerlendirilmesi

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

Davranış

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.

Yüzme felç indüklenen Caenorhabditis elegans dopamin sinyal değerlendirmek için

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.

Parkinson Hastalığında Maskeyi Düşürme Duyarlılık Mekanizmalarına Gen-Çevre Etkileşim Modelleri

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

Tıp

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.

Içinde Nörodejeneratif fenotipleri değerlendirilmesi<em&gt; Drosophila</em&gt; Tırmanma Tahliller tarafından Dopaminerjik nöronlar ve Tüm Beyin immün

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

Nörobilim

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.

Fare dopaminerjik nöronlar İlköğretim Kültür

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.

Nörodejeneratif Hastalıkların bir Drosophila Modelinde Tırmanma Kusur Kantitatif Analiz

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.

RNA sıralaması ve TH-organizatörü odaklı eGFP İfade Kullanımı orta beyin Kültürlerde Yaşayan dopaminerjik nöronlar güvenilir Kimlik

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.

Parkinson Hastalığının Genetik Sıçan Modelinde Yürüme Analizi için RatWalker Sisteminin Uygulanması

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.

Caenorhabditis elegans'ta Dopaminerjik Nöron Morfolojik Değişim ve Dejenerasyon Seviyelerinin Ölçülmesi

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

Fenotipik Profilleme için İnsan Orta Beyin Dopaminerjik Nöronlarının Kültürlenmesi

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.

Yazar Spotlight: Nöronal Fenotipik Profiller Oluşturma - İnsan Orta Beyin Dopaminerjik Nöronlarını Kültür ve İmaj İçin Bir Protokol 

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.

İnsan Kök Hücre Kaynaklı Orta Beyin Dopaminerjik Nöronlarının Fenotipik Profillemesi

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 ...