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1.
Brain Commun ; 3(2): fcab105, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34131644

RESUMO

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and 'gatepoints' to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 µm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10-15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal.

2.
Brain Commun ; 2(2): fcaa066, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32954323

RESUMO

Huntington's disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington's disease, that were known to abrogate somatic instability in Huntington's disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington's disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington's disease patient's blood, but which has been detected in post-mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)90 mice and that this correlated with the onset of transcriptional dysregulation. Whereas in R6/2(CAG)200 mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)90 brains have different properties to those in the R6/2(CAG)200 mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilized by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)200 brains polymerize into larger structures that appear as inclusion bodies. These data emphasize that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.

3.
Int J Neuropsychopharmacol ; 12(2): 267-73, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19149910

RESUMO

Chromosome 15q13-q14 harbours the gene for the alpha7 nicotinic acetylcholine receptor subunit (CHRNA7) and a related gene (CHRFAM7A) which arises from a partly duplicated portion of CHRNA7. Recent evidence suggests that CHRFAM7A is a locus with a possible role in schizophrenia and cognitive functioning. We studied an antisaccade task as a fronto-parietal measure of executive function that reflects risk for schizophrenia. Association of CHRFAM7A genotype with antisaccade performance was assessed in 103 healthy Caucasian individuals. No significant associations of 2-bp deletion or CHRFAM7A copy number with antisaccade performance parameters were observed. The failure to observe an association between antisaccade performance and polymorphisms in CHRFAM7A gene is consistent with specificity of the gene effects on hippocampal and memory functions as previously demonstrated.


Assuntos
Polimorfismo Genético/genética , Resolução de Problemas/fisiologia , Receptores Nicotínicos/genética , Movimentos Sacádicos/genética , Deleção de Sequência/genética , Adulto , Análise de Variância , Eletroculografia/métodos , Feminino , Genótipo , Humanos , Masculino , Personalidade/genética , Inquéritos e Questionários , Adulto Jovem , Receptor Nicotínico de Acetilcolina alfa7
4.
Eur J Hum Genet ; 16(11): 1364-71, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18545269

RESUMO

The alpha7 nicotinic acetylcholine receptor gene (CHRNA7) is located at 15q13-q14 in a region that is strongly linked to the P50 sensory gating deficit, an endophenotype of schizophrenia and bipolar disorder. Part of the gene is a copy number variant, due to a duplication of exons 5-10 and 3' sequence in CHRFAM7A, which is present in many but not all humans. Maps of this region show that the two genes are in opposite orientation in the individual mainly represented in the public access human DNA sequence database (Build 36), suggesting that an inversion had occurred since the duplication. We have used fluorescent in situ hybridization to investigate this putative inversion. Analysis of interphase chromosomes in 12 individuals confirms the occurrence of an inversion and indicates that CHRFAM7A exists in both orientations with similar frequency. We showed that the 2 bp deletion polymorphism in exon 6 of CHRFAM7A is in strong linkage disequilibrium with the inversion polymorphism (r(2)=0.82, CI 0.53-1.00, P=0.00003), which can therefore be used as a surrogate marker. Previous associations of endophenotypes of schizophrenia with the 2 bp deletion might therefore be due to the orientation of the duplicon containing CHRFAM7A.


Assuntos
Sequência de Bases/genética , Transtorno Bipolar/genética , Inversão Cromossômica , Cromossomos Humanos Par 15/genética , Receptores Nicotínicos/genética , Esquizofrenia/genética , Deleção de Sequência , Animais , Mapeamento Cromossômico , Feminino , Marcadores Genéticos/genética , Humanos , Desequilíbrio de Ligação , Masculino , Pan troglodytes , Polimorfismo Genético , Receptor Nicotínico de Acetilcolina alfa7
5.
Nucleic Acids Res ; 32(7): 2113-22, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15087490

RESUMO

Adenosine to inosine editing of mRNA from the human 5-HT2C receptor gene (HTR2C) occurs at five exonic positions (A-E) in a stable stem-loop that includes the normal 5' splice site of intron 5 and is flanked by two alternative splice sites. Using in vitro editing, we identified a novel editing site (F) located in the intronic part of the stem-loop and demonstrated editing at this site in human brain. We have shown that in cell culture, base substitutions to mimic editing at different combinations of the six sites profoundly affect relative splicing at the normal and the upstream alternative splice site, but splicing at the downstream alternative splice site was consistently rare. Editing combinations in different splice variants from human brain were determined and are consistent with the effects of editing on splicing observed in cell culture. As RNA editing usually occurs close to exon/intron boundaries, this is likely to be a general phenomenon and suggests an important novel role for RNA editing.


Assuntos
Processamento Alternativo/genética , Edição de RNA , Receptor 5-HT2C de Serotonina/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Sítios de Ligação/genética , Encéfalo/metabolismo , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Íntrons/genética , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Células PC12 , RNA/genética , RNA/metabolismo , Ratos
6.
Eur J Hum Genet ; 21(1): 76-81, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22588665

RESUMO

There is considerable evidence implicating the 15q13.3 region in neuropsychiatric disorders, with the α7 nicotinic receptor gene CHRNA7 the most plausible candidate. This region has multiple duplications and many copy number variants (CNVs). A common CNV involves a partial duplication of CHRNA7 (CHRFAM7A), which occurs in either orientation. We examined the distribution of these alternative genomic arrangements in a large cohort of psychiatric patients, their relatives and controls using the 2-bp deletion polymorphism as a marker for the orientation of CHRFAM7A. We investigated three common alleles for association with psychosis and with the P50 sensory gating deficit, which is strongly associated with psychosis and strongly linked to 15q13.3. We found significant within-family association with P50 (empirical P=0.004), which is robust to population stratification. Most of the effect came from the 2-bp deletion allele, which tags the variant of CHRFAM7A in the same orientation as CHRNA7. This allele is associated with the presence of the P50 sensory gating deficit (empirical P=0.0006). Tests comparing within-family and between-family components of association suggest considerable population stratification in the sample. We found no evidence for association with psychosis, but this may reflect lower power using this phenotype. Four out of six previous association studies found association of different psychiatric phenotypes with the same 2-bp deletion allele.


Assuntos
Polimorfismo Genético , Transtornos Psicóticos/genética , Receptores Nicotínicos/genética , Filtro Sensorial/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Deleção Cromossômica , Cromossomos Humanos Par 15 , Doenças em Gêmeos/genética , Eletroencefalografia , Feminino , Duplicação Gênica , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Nicotínicos/metabolismo , População Branca/genética , Adulto Jovem , Receptor Nicotínico de Acetilcolina alfa7
7.
Neurosci Lett ; 497(2): 139-43, 2011 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-21569822

RESUMO

The astroglial EAAT2 glutamate transporter is essential for clearing glutamate in the central nervous system and protecting against excitotoxicity. It is implicated in amyotrophic lateral sclerosis (ALS, the most common type of motor neurone disease) where less EAAT2 is found, possibly involving aberrant intron 7 retention transcripts. We report adenine/inosine RNA editing at a novel site in intron 7 of EAAT2 pre-mRNA that appears to activate a cryptic alternative polyadenylation site, generating intron 7 retention transcripts. This polyadenylation site includes two overlapping polyadenylation signals opposite the editing site in a strong stem-loop, which is highly conserved in primates. In pre-mRNA, we observed variable editing levels at this site, which were significantly higher in spinal cord (p=0.001) and motor cortex (p=0.005) from ALS patients, but not in cerebellum, demonstrating specificity for clinically relevant regions. By contrast, incomplete mRNA molecules polyadenylated in intron 7 are always completely edited. Cell culture experiments confirm this strong correlation between editing and polyadenylation in intron 7, strongly suggesting activation of the alternative polyadenylation site by editing. Prediction of inosine base-pairing from published data suggests that RNA editing releases the polyadenylation signals from the stem-loop, providing a plausible mechanism. To the best of our knowledge, this is the first report of RNA editing activating an alternative polyadenylation signal.


Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Transporte de Glutamato da Membrana Plasmática/genética , Poliadenilação/genética , Edição de RNA/genética , Precursores de RNA/genética , Regulação para Cima/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Linhagem Celular Tumoral , Transportador 2 de Aminoácido Excitatório , Gorilla gorilla , Humanos , Hylobates , Córtex Motor/patologia , Pan troglodytes , Sítios de Splice de RNA/genética , Medula Espinal/patologia
8.
Genome Biol ; 8(6): R114, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17573966

RESUMO

BACKGROUND: Chromosome 15 contains many segmental duplications, including some at 15q11-q13 that appear to be responsible for the deletions that cause Prader-Willi and Angelman syndromes and for other genomic disorders. The current version of the human genome sequence is incomplete, with seven gaps in the proximal region of 15q, some of which are flanked by duplicated sequence. We have investigated this region by conducting a detailed examination of the sequenced genomic clones in the public database, focusing on clones from the RP11 library that originates from one individual. RESULTS: Our analysis has revealed assembly errors, including contig NT_078094 being in the wrong orientation, and has enabled most of the gaps between contigs to be closed. We have constructed a map in which segmental duplications are no longer interrupted by gaps and which together reveals a complex region. There are two pairs of large direct repeats that are located in regions consistent with the two classes of deletions associated with Prader-Willi and Angelman syndromes. There are also large inverted repeats that account for the formation of the observed supernumerary marker chromosomes containing two copies of the proximal end of 15q and associated with autism spectrum disorders when involving duplications of maternal origin (inv dup[15] syndrome). CONCLUSION: We have produced a segmental map of 15q11-q14 that reveals several large direct and inverted repeats that are incompletely and inaccurately represented on the current human genome sequence. Some of these repeats are clearly responsible for deletions and duplications in known genomic disorders, whereas some may increase susceptibility to other disorders.


Assuntos
Síndrome de Angelman/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 15 , Síndrome de Prader-Willi/genética , Quebra Cromossômica , Duplicação Gênica , Genoma Humano , Humanos
9.
Am J Med Genet B Neuropsychiatr Genet ; 141B(6): 571-5, 2006 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-16823804

RESUMO

Schizophrenia and bipolar disorder are major psychiatric diseases that have a strong genetic element. Markers in the vicinity of the CHRNA7 gene at 15q13-q14 have been linked with an endophenotype of schizophrenia, P50 sensory gating disorder, with schizophrenia itself and with bipolar disorder. We have measured the copy number of the polymorphic partial duplication of CHRNA7 (CHRFAM7A) and genotyped a polymorphic 2 bp deletion within exon 6 of CHRFAM7A. In this study, 208 probands with a primary diagnosis of schizophrenia, 217 with a diagnosis of bipolar affective disorder and 28 with schizoaffective or other psychotic disorders were examined together with 197 controls recruited from the same region in Scotland. No significant association was seen for schizophrenia and bipolar disorder by genotype or allele overall for either polymorphism, but a mildly significant association by genotype (P = 0.04) was observed for absence of CHRFAM7A when the sample was analyzed as a single psychosis phenotype.


Assuntos
Transtorno Bipolar/genética , Polimorfismo Genético , Receptores Nicotínicos/genética , Esquizofrenia/genética , Sequência de Bases , Cromossomos Humanos Par 15 , Primers do DNA , Humanos , Receptor Nicotínico de Acetilcolina alfa7
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