Name: a scalable, cell-based method for the functional assessment of ube3a variants
Uploaded: 2022-10-10T15:00:00
Description: Watch this Scientific Journal Video about A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants at JoVE.com

Este trabalho foi apoiado por um Simons Foundation Bridge to Independence Award (SFARI Award #387972; J.J.Y.), um NARSAD Young Investigator Award da Brain and Behavior Research Foundation (J.J.Y.), uma bolsa de pesquisa da Alfred P. Sloan Foundation (J.J.Y.), e bolsas de pesquisa da Angelman Syndrome Foundation (J.J.Y.), da Whitehall Foundation (J.J.Y.) e do NIMH (R01MH122786; J.J.Y.).

1. Clonagem de mutagênese para gerar variantes do Ube3a
<ol><li>Clone todas as variantes de Ube3a no plasmídeo pCIG2 (Figura 1A), um vetor bicistrônico que contém um promotor de acetina β galinha e um local de entrada interno de ribossomo (IRES) para a expressão EGFP19. Construções Ube3a de comprimento total contêm uma sequência de N-terminal Myc-tag e são clonadas em pCIG2 usando um site SacI de 5' e um site Xm...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+interpretation:+Functional+assays+to+the+rescue.">Variant interpretation: Functional assays to the rescue.</a> American Journal of Human Genetics. 101 (3), 315-325 (2017).</li><li>Scheffner, M., Huibregtse, J. M., Vierstra, R. D., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HPV-16+E6+and+E6-AP+complex+functions+as+a+ubiquitin-protein+ligase+in+the+ubiquitination+of+p53.">The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.</a> Cell. 75 (3), 495-505 (1993).</li><li>Albrecht, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+expression+of+the+murine+Angelman+syndrome+gene,+Ube3a,+in+hippocampal+and+Purkinje+neurons.">Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.</a> Nature Genetics. 17 (1), 75-78 (1997).</li><li>Rougeulle, C., Glatt, H., Lalande, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Angelman+syndrome+candidate+gene,+UBE3A/E6-AP,+is+imprinted+in+brain.">The Angelman syndrome candidate gene, UBE3A/E6-AP, is imprinted in brain.</a> Nature Genetics. 17 (1), 14-15 (1997).</li><li>Vu, T. H., Hoffman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinting+of+the+Angelman+syndrome+gene,+UBE3A,+is+restricted+to+brain.">Imprinting of the Angelman syndrome gene, UBE3A, is restricted to brain.</a> Nature Genetics. 17 (1), 12-13 (1997).</li><li>Kishino, T., Lalande, M., Wagstaff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UBE3A/E6-AP+mutations+cause+Angelman+syndrome.">UBE3A/E6-AP mutations cause Angelman syndrome.</a> Nature Genetics. 15 (1), 70-73 (1997).</li><li>Jiang, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+of+the+Angelman+ubiquitin+ligase+in+mice+causes+increased+cytoplasmic+p53+and+deficits+of+contextual+learning+and+long-term+potentiation.">Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.</a> Neuron. 21 (4), 799-811 (1998).</li><li>Mabb, A. M., Judson, M. C., Zylka, M. J., Philpot, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angelman+syndrome:+Insights+into+genomic+imprinting+and+neurodevelopmental+phenotypes.">Angelman syndrome: Insights into genomic imprinting and neurodevelopmental phenotypes.</a> Trends in Neuroscience. 34 (6), 293-303 (2011).</li><li>Hogart, A., Wu, D., LaSalle, J. M., Schanen, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comorbidity+of+autism+with+the+genomic+disorders+of+chromosome+15q11.2-q13.">The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13.</a> Neurobiology of Disease. 38 (2), 181-191 (2010).</li><li>Urraca, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interstitial+duplication+15q11.2-q13+syndrome+includes+autism,+mild+facial+anomalies+and+a+characteristic+EEG+signature.">The interstitial duplication 15q11.2-q13 syndrome includes autism, mild facial anomalies and a characteristic EEG signature.</a> Autism Research. 6 (4), 268-279 (2013).</li><li>de la Torre-Ubieta, L., Won, H., Stein, J. L., Geschwind, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+Angelman+syndrome-associated+mutations+in+the+E3+ubiquitin+ligase+E6-associated+protein.">Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein.</a> Journal of Biological Chemistry. 279 (39), 41208-41217 (2004).</li><li>Yi, J. J., Barnes, A. P., Hand, R., Polleux, F., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+specifies+axons+during+brain+development.">TGF-beta signaling specifies axons during brain development.</a> Cell. 142 (1), 144-157 (2010).</li><li>Yi, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autism-linked+UBE3A+T485A+mutant+E3+ubiquitin+ligase+activates+the+Wnt/beta-catenin+pathway+by+inhibiting+the+proteasome.">The autism-linked UBE3A T485A mutant E3 ubiquitin ligase activates the Wnt/beta-catenin pathway by inhibiting the proteasome.</a> Journal of Biological Chemistry. 292 (30), 12503-12515 (2017).</li><li>Weston, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+disease-linked+hyperactivating+mutations+in+UBE3A+through+large-scale+functional+variant+analysis.">Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis.</a> Nature Communications. 12 (1), 6809 (2021).</li><li>Hand, R., Polleux, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin2+regulates+the+initial+axon+guidance+of+cortical+pyramidal+neurons+projecting+medially+to+the+corpus+callosum.">Neurogenin2 regulates the initial axon guidance of cortical pyramidal neurons projecting medially to the corpus callosum.</a> Neural Development. 6, 30 (2011).</li><li>Karginov, A. V., Ding, F., Kota, P., Dokholyan, N. V., Hahn, K. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Autism-linked+mutation+disables+phosphorylation+control+of+UBE3A.">An Autism-linked mutation disables phosphorylation control of UBE3A.</a> Cell. 162 (4), 795-807 (2015).</li><li>Kuhnle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angelman+syndrome-associated+point+mutations+in+the+Zn(2+)-binding+N-terminal+(AZUL)+domain+of+UBE3A+ubiquitin+ligase+inhibit+binding+to+the+proteasome.">Angelman syndrome-associated point mutations in the Zn(2+)-binding N-terminal (AZUL) domain of UBE3A ubiquitin ligase inhibit binding to the proteasome.</a> Journal of Biological Chemistry. 293 (47), 18387-18399 (2018).</li><li>Yamamoto, Y., Huibregtse, J. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+E6-AP+gene+(UBE3A)+encodes+three+potential+protein+isoforms+generated+by+differential+splicing.">The human E6-AP gene (UBE3A) encodes three potential protein isoforms generated by differential splicing.</a> Genomics. 41 (2), 263-266 (1997).</li><li>Avagliano Trezza, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+nuclear+UBE3A+causes+electrophysiological+and+behavioral+deficits+in+mice+and+is+associated+with+Angelman+syndrome.">Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.</a> Nature Neuroscience. 22 (8), 1235-1247 (2019).</li><li>Bossuyt, S. N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+nuclear+UBE3A+activity+is+the+predominant+cause+of+Angelman+syndrome+in+individuals+carrying+UBE3A+missense+mutations.">Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations.</a> Human Molecular Genetics. 30 (6), 430-442 (2021).</li></ol>

O protocolo aqui descrito fornece um método eficiente e escalável para avaliar a atividade enzimática das variantes de Ube3a. Existem vários detalhes técnicos que merecem uma consideração cuidadosa ao usar este ensaio. Uma consideração é a escolha dos plasmídeos repórteres Wnt utilizados neste ensaio. O protocolo descrito aqui usa especificamente o β-catenin activated reporter (BAR)21, um reportador que contém um concatenador de 12 elementos de resposta do fator de células ...

Avanços tecnológicos recentes têm tornado rotineiro o sequenciamento de exomas e genomas em ambientes clínicos 1,2. Isso levou à descoberta de um grande número de variantes genéticas, incluindo milhões de variantes missense que normalmente mudam um aminoácido em uma proteína. Compreender o significado funcional dessas variantes continua sendo um desafio, e apenas uma pequena fração (~2%) das variantes de missense conhecidas tem uma interpretação clínica 1,3.
Um exemplo proeminente desse problema é o Ube3a, um gene que codifica uma ligase de ubiquitina E3 que tem como alvo proteínas de substrato para degradação4. Ube3a reside no cromossomo 15q11-13 e é expresso exclusivamente a partir do alelo herdado maternalmente 5,6,7. Observações da genética da doença sugerem fortemente que a atividade insuficiente ou excessiva da enzima UBE3A causa distúrbios distintos do neurodesenvolvimento. A deleção do cromossomo materno 15q11-13 causa síndrome de Angelman (EA)8, distúrbio caracterizado por deficiência intelectual grave, comprometimento motor, convulsões, comportamento feliz com sorriso frequente e características faciais dismórficas 8,9,10. Em contraste, a duplicação ou triplicação do cromossomo materno 15q11-13 causa a síndrome de Dup15q, uma condição heterogênea reconhecida como uma das formas sindrômicas mais prevalentes de autismo11,12,13. Além disso, existem centenas de variantes missense identificadas no Ube3a, a maioria das quais são consideradas variantes de significado incerto (USV), pois seu significado funcional e clínico são desconhecidos. Assim, há um interesse considerável no desenvolvimento de métodos que possam avaliar empiricamente as variantes do Ube3a para determinar se elas contribuem para a doença do neurodesenvolvimento.
A enzima UBE3A pertence à família de domínios HECT (homóloga ao E6-AP C-terminus) de ligases E3 ubiquitina que possuem o domínio homônimo HECT, que contém o maquinário bioquímico necessário para aceitar ubiquitina ativada de enzimas E2 e transferi-la para proteínas de substrato14. Historicamente, a caracterização das enzimas E3 tem se baseado em sistemas in vitro reconstituídos que requerem um conjunto de proteínas purificadas 4,15,16. Tais métodos são lentos e trabalhosos e não são passíveis de avaliar a atividade de um grande número de variantes. Em trabalhos anteriores, o UBE3A foi identificado para ativar a via Wnt em células HEK293T, modulando a função do proteassoma para retardar a degradação da β-catenina17. Essa percepção permite o uso de repórteres de via Wnt como um método eficiente e rápido para identificar variantes de perda e ganho de função do Ube3a18. O protocolo abaixo descreve em detalhes um método para gerar variantes Ube3a, bem como um repórter baseado em luciferase para avaliar mudanças na atividade de variantes Ube3a.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA (1x), phenol red</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>1 Kb DNA ladder</td>
			<td>Lambda Biotech</td>
			<td>M108-S</td>
			<td></td>
		</tr>
		<tr>
			<td>100 bp DNA Ladder</td>
			<td>Lambda Biotech</td>
			<td>M107</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Buffer for T4 DNA Ligase with 10 mM ATP</td>
			<td>New England BioLabs</td>
			<td>B0202A</td>
			<td></td>
		</tr>
		<tr>
			<td>5x Phusion HF Reaction Buffer</td>
			<td>New England BioLabs</td>
			<td>B0518S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic Solution</td>
			<td>Corning</td>
			<td>30004CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Black/White Isoplate-96 Black Frame White Well plate</td>
			<td>PerkinElmer</td>
			<td>6005030</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin Disodium Salt</td>
			<td>Midwest Scientific</td>
			<td>KCC46000-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber&#160; slides</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II Automated Cell Counter&#160;</td>
			<td>life technologies</td>
			<td></td>
			<td>Cell counting machine</td>
		</tr>
		<tr>
			<td>Custom DNA oligos</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Mix</td>
			<td>New England BioLabs</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569044</td>
			<td>Basal medium for supporting the growth of HEK293T cell line</td>
		</tr>
		<tr>
			<td>DPBS (1x)</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1910</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI-HF&#160;</td>
			<td>New England BioLabs</td>
			<td>R3101S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr>
			<td>Fisherbrand Surface Treated Tissue Culture Dishes</td>
			<td>Fisherbrand</td>
			<td>FB012924</td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2691</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye Purple (6x)</td>
			<td>New England BioLabs</td>
			<td>B7024A</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High Efficiency ig 10B Chemically Competent Cells</td>
			<td>Intact Genomics</td>
			<td>1011-12</td>
			<td>E. coli DH10B cells</td>
		</tr>
		<tr>
			<td>HiSpeed Plasmid Midi Kit</td>
			<td>Qiagen</td>
			<td>12643</td>
			<td>Midi prep</td>
		</tr>
		<tr>
			<td>pCIG2 plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pGL3 BAR plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion HF DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0530L</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>ProFlex 3 x 32 well PCR System</td>
			<td>Applied biosystems by life technologies</td>
			<td></td>
			<td>Thermocycler</td>
		</tr>
		<tr>
			<td>pTK Renilla plasmid</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>Mini prep</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (250)</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td>Gel purification</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit (250)</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>rCutSmart Buffer</td>
			<td>New England BioLabs</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>SacI-HF</td>
			<td>New England BioLabs</td>
			<td>R3156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Synergy HTX Multi-Mode Reader</td>
			<td>BioTek</td>
			<td></td>
			<td>&#160;Plate reader runs Gen5 software v3.08 (BioTek)</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England BioLabs</td>
			<td>M0202L</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>TAE Buffer, Tris-Acetate-EDTA, 50x Solution, Electrophoresis</td>
			<td>Fisher Scientific</td>
			<td>BP13324</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 96 wells, Flat Bottom</td>
			<td>Fisherbrand</td>
			<td>FB012931</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Ethidium Bromide Solution</td>
			<td>Invitrogen by Thermo Fisher Scientific</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>XmaI</td>
			<td>New England BioLabs</td>
			<td>R0180S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

a scalable, cell-based method for the functional assessment of ube3a variants

O aumento do uso de sequenciamento na medicina identificou milhões de variantes codificadoras no genoma humano. Muitas dessas variantes ocorrem em genes associados a distúrbios do neurodesenvolvimento, mas o significado funcional da grande maioria das variantes permanece desconhecido. O presente protocolo descreve o estudo de variantes para Ube3a, um gene que codifica uma ubiquitina ligase E3 ligada ao autismo e à síndrome de Angelman. A duplicação ou triplicação de Ube3a está fortemente ligada ao autismo, enquanto sua deleção causa a síndrome de Angelman. Assim, a compreensão da valência das alterações na atividade da proteína UBE3A é importante para os desfechos clínicos. Aqui, um método rápido, baseado em células, que emparelha variantes Ube3a com um repórter de via Wnt é descrito. Este ensaio simples é escalável e pode ser usado para determinar a valência e a magnitude das mudanças de atividade em qualquer variante do Ube3a . Além disso, a facilidade deste método permite a geração de uma riqueza de informações estrutura-função, que podem ser usadas para obter insights profundos sobre os mecanismos enzimáticos da UBE3A.

A triagem funcional em larga escala de variantes de Missense do Ube3a identifica um amplo cenário de mutações de perda e ganho de função Trabalhos anteriores com mutantes Ube3a sugeriram que a resposta Wnt pode servir como um repórter da atividade celular da proteína UBE3A. Essas observações foram expandidas e experimentos de validação adicionais foram realizados para investigar se o ensaio BAR é adequado para relatar uma série de atividades UBE3A na célula. Primeiro, a...

Watch this Scientific Journal Video about A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants at JoVE.com

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Um método simples e escalável foi desenvolvido para avaliar o significado funcional de variantes missense em Ube3a, um gene cuja perda e ganho de função estão ligados à síndrome de Angelman e ao transtorno do espectro do autismo.

Um método escalável baseado em células para a avaliação funcional de variantes do Ube3a

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

Nosso método fornece uma maneira rápida de entender o impacto funcional das variantes em Ube3a, uma ubiquitina ligase que causa a síndrome de Angelman e está fortemente ligada a uma forma genética de autismo conhecida como síndrome de Dup15q. A caracterização da atividade da ubiquitina ligase é tradicionalmente trabalhosa e lenta, e tais métodos são difíceis e não realmente passíveis de caracterizar as centenas de variantes identificadas para Ube3a. A síndrome de Angelman é causada pela deleção do gene Ube3a materno, enquanto a síndrome Dup15q é causada pela duplicação do Ube3a materno, por isso é importante deduzir como variantes missense mudaram a atividade da enzima Ube3a, e isso é algo que é difícil de fazer sem testar empiricamente cada variante.Demonstrando o procedimento estará Jalin Stelzer, um técnico de pesquisa no meu laboratório. Comece realizando os ensaios em células HEK293T, cultivadas no Meio Águia Modificado de Dulbecco a 37 graus Celsius, com 5% de dióxido de carbono em um recipiente umidificado, conforme descrito no manuscrito. Em um gabinete de biossegurança, coloque as células HEK293T suspensas em uma placa de fundo plano 96 bem tratada com cultura de tecidos e incube as células durante a noite a 37 graus.No segundo dia, transfecte as células em um gabinete de biossegurança com plasmídeos repórteres Firefly e Renilla luciferase e plasmídeos Ube3a em triplicado. Realizar as transfecções em um volume total de 10 microlitros por poço. Crie uma mistura mestra para as transfecções para cada variante em um tubo de microcentrífuga de 1,5 mililitro e incube à temperatura ambiente por 15 minutos.Após a incubação, agite suavemente a mistura de transfecção batendo no tubo e, em seguida, adicione 10 microlitros da mistura de transfecção diretamente ao meio de crescimento existente nos poços. Depois, devolva as células à incubadora e permita que os plasmídeos se expressem por 48 horas. Usando um microscópio de fluorescência, monitore a eficiência da transfecção por fluorescência GFP.Mais de 80% das células devem expressar GFP após 48 horas. Aspirar cuidadosamente os meios de cultura das células HEK293T transfectadas e, em seguida, lavar as células com PBS frio. Aspirar o PBS.Adicione 25 microlitros de tampão de lise não desnaturante para lise celular e incube por 15 minutos no gelo com balanço suave. Em seguida, adicione 20 microlitros do lisado resultante em uma nova placa de ensaio de 96 poços contendo 100 microlitros de um reagente de substrato de luciferase Firefly e misture a placa suavemente tocando suavemente. Carregue a placa até um leitor de placas e meça a luminescência usando um detector superior com uma altura de leitura de um milímetro e um tempo de integração de um segundo.Imediatamente após a leitura da luminescência da luciferase Firefly, adicione 100 microlitros de substrato de luciferase Renilla aos poços, o que extingue a atividade da luciferase Firefly enquanto avalia a atividade da luciferase Renilla. Misture as amostras por agitação suave da placa e meça a luminescência novamente para avaliar a atividade da luciferase de Renilla. Compare a atividade relativa das proteínas variantes quantificando as respostas do repórter e normalizando o valor de Firefly e Renilla para cada variante em relação ao tipo selvagem Ube3a.As células HEK293T transfectadas com quantidades crescentes de plasmídeos que codificam Ube3a do tipo selvagem mostram um aumento linear na resposta BAR. O ensaio BAR identifica variantes dos plasmídeos ligadas à doença, demonstrando a precisão e a generalidade deste método. A tela de ensaio BAR para 152 variantes de Ube3a exibe perda benigna de função e ganho de mutações de função em relação ao Ube3a do tipo selvagem.O ganho de função de substituição de glutamato, perda de função de substituição de prolina e outra perda de função substituição de arginina na posição 588 de Ube3a produziram efeitos drasticamente diferentes, ressaltando assim a importância da avaliação empírica de variantes. O gráfico de calor mostra resultados normalizados de BAR para análise mutacional abrangente na posição 588 em Ube3a. O sombreamento branco representa os níveis de atividade Ube3a do tipo selvagem, o sombreamento azul indica perda de função e o sombreamento vermelho indica ganho de mutações de função.Além das implicações clínicas do nosso trabalho, o Ube3a é um membro da família de ligases ubiquitina do domínio HECT, que possuem o domínio catalítico HECT conservado. E eu acho que um dos aspectos fascinantes do nosso trabalho é que ele fornece dados ricos de função de estrutura sobre Ube3a, e isso pode ser usado para obter uma compreensão mecanicista mais profunda para esta classe de enzimas.

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Neuroscience

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

Escalável Fluidic Arrays Injector para Viral Segmentação de Intact Circuitos 3-D do cérebro

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

Neurociência

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

Mapeamento funcional com MEG e EEG em simultâneo

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

Avaliação da lateralização cerebral em crianças usando Funcional ultra-som Doppler transcraniano (fTCD)

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

De alta resolução funcionais Métodos ressonância magnética para Midbrain Humanos

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Funcional e morfológica de Avaliação de Diafragma Innervation por frênico Neurônios Motores

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Avaliação da complexidade dendrítica do hipocampo em ratos idosos usando o método de Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: um novo método para Fast Neurite Extension e Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Um método volumétrico para quantificação de vasoespasmo Cerebral em um modelo murino de hemorragia subaracnoide

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

O poder do intervalo de Interstimulus para a avaliação do processamento Temporal em roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

&#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in the taste sensory organ. By integrating microfluidics into the intravital tongue imaging window, the &#181;Tongue provides reliable functional images of taste cells in vivo under controlled exposure to multiple tastants. In this paper, a detailed step-by-step procedure to utilize the &#181;Tongue system is presented. There are five subsections: preparing of tastant solutions, setting up of a microfluidic module, sample mounting, acquiring functional image data, and data analysis. Some tips and techniques to solve the practical issues that may arise when using the &#181;Tongue are also presented.

 &#181;Tongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

The article introduces the &#181;Tongue (microfluidics-on-a-tongue) device for functional taste cell imaging in vivo by integrating microfluidics into an intravital imaging window on the tongue.

μTongue: Uma plataforma de imagem funcional baseada em microfluidics para a língua in vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...