RESUMO
The incidence of many cancer types is significantly reduced in individuals with Down's syndrome, and it is thought that this broad cancer protection is conferred by the increased expression of one or more of the 231 supernumerary genes on the extra copy of chromosome 21. One such gene is Down's syndrome candidate region-1 (DSCR1, also known as RCAN1), which encodes a protein that suppresses vascular endothelial growth factor (VEGF)-mediated angiogenic signalling by the calcineurin pathway. Here we show that DSCR1 is increased in Down's syndrome tissues and in a mouse model of Down's syndrome. Furthermore, we show that the modest increase in expression afforded by a single extra transgenic copy of Dscr1 is sufficient to confer significant suppression of tumour growth in mice, and that such resistance is a consequence of a deficit in tumour angiogenesis arising from suppression of the calcineurin pathway. We also provide evidence that attenuation of calcineurin activity by DSCR1, together with another chromosome 21 gene Dyrk1a, may be sufficient to markedly diminish angiogenesis. These data provide a mechanism for the reduced cancer incidence in Down's syndrome and identify the calcineurin signalling pathway, and its regulators DSCR1 and DYRK1A, as potential therapeutic targets in cancers arising in all individuals.
Assuntos
Síndrome de Down/genética , Inositol/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Animais , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio , Catecóis , Células Cultivadas , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Síndrome de Down/metabolismo , Células Endoteliais/metabolismo , Dosagem de Genes/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases DyrkRESUMO
Down syndrome (DS) is the leading non-heritable cause of mental retardation and is due to the effects of an extra chromosome 21. Mouse models of DS have been developed which parallel many of the cognitive and behavioral deficits of DS individuals. Of these, Ts65Dn mice show abnormal hippocampal properties including learning and memory deficits, altered synaptic plasticity and irregular dendritic spines. We assessed synaptic function of cultured postnatal Ts65Dn hippocampal neurons through examination of spontaneous miniature excitatory post-synaptic currents (mEPSCs) and compared them to those from diploid neurons. Averaged amplitudes and frequency of mEPSC events were similar to diploid suggesting presynaptic function is not overtly disrupted in Ts65Dn hippocampal neurons. However, both averaged decay and rise times (10-90% of peak) were significantly faster (approximately 20% for both rise and decay) in Ts65Dn neurons compared to diploid. The distribution of both decay and rise times, indicates global scaling of all percentile groups and is independent of amplitude suggesting normal electrotonic filtering in spite of abnormal expression of GIRK2 channel in Ts65Dn mouse. Western blot analysis suggests overexpression of GluR4 subunit of AMPA receptors which may contribute to faster mEPSC in Ts65Dn neurons. Intrinsic synaptic properties influenced by genetics or epigenetics factors in Ts65Dn postnatal cultured neurons are therefore disrupted and may contribute to the cognitive deficits associated with DS.
Assuntos
Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/citologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Modelos Animais de Doenças , Síndrome de Down/genética , Síndrome de Down/patologia , Potenciais Pós-Sinápticos Excitadores/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , Tempo de Reação/fisiologia , Tempo de Reação/efeitos da radiação , Receptores de AMPA/metabolismoRESUMO
Ts65Dn, a mouse model of Down syndrome (DS), demonstrates abnormal hippocampal synaptic plasticity and behavioral abnormalities related to spatial learning and memory. The molecular mechanisms leading to these impairments have not been identified. In this study, we focused on the G-protein-activated inwardly rectifying potassium channel 2 (GIRK2) gene that is highly expressed in the hippocampus region. We studied the expression pattern of GIRK subunits in Ts65Dn and found that GIRK2 was overexpressed in all analyzed Ts65Dn brain regions. Interestingly, elevated levels of GIRK2 protein in the Ts65Dn hippocampus and frontal cortex correlated with elevated levels of GIRK1 protein. This suggests that heteromeric GIRK1-GIRK2 channels are overexpressed in Ts65Dn hippocampus and frontal cortex, which could impair excitatory input and modulate spike frequency and synaptic kinetics in the affected regions. All GIRK2 splicing isoforms examined were expressed at higher levels in the Ts65Dn in comparison to the diploid hippocampus. The pattern of GIRK2 expression in the Ts65Dn mouse brain revealed by in situ hybridization and immunohistochemistry was similar to that previously reported in the rodent brain. However, in the Ts65Dn mouse a strong immunofluorescent staining of GIRK2 was detected in the lacunosum molecular layer of the CA3 area of the hippocampus. In addition, tyrosine hydroxylase containing dopaminergic neurons that coexpress GIRK2 were more numerous in the substantia nigra compacta and ventral tegmental area in the Ts65Dn compared to diploid controls. In summary, the regional localization and the increased brain levels coupled with known function of the GIRK channel may suggest an important contribution of GIRK2 containing channels to Ts65Dn and thus to DS neurophysiological phenotypes.
Assuntos
Síndrome de Down/metabolismo , Lobo Frontal/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Hipocampo/metabolismo , Substância Negra/metabolismo , Animais , Modelos Animais de Doenças , Síndrome de Down/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Regulação da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não ParamétricasRESUMO
Due to the homology between human chromosome 21 and mouse chromosome 16, trisomy 16 mice are considered animal models of Down syndrome (DS). Abnormal hippocampal synaptic plasticity and behavior have been reported in the segmental trisomy 16 Ts65Dn mouse. In the Ts1Cje DS mouse model, which has a shorter triplicated chromosomal segment than Ts65Dn, more subtle hippocampal behavioral deficits have been reported. In this study, we investigated CA1 hippocampal synaptic plasticity, long-term potentiation (LTP) and depression (LTD) in the Ts1Cje mouse. Field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 area of in vitro hippocampal slices from the Ts1Cje mouse and diploid controls, LTP was induced by a single tetanizing train pulse (1 s) at 100 Hz and LTD by a 900-pulse train at 1 Hz. We report for the first time that compared to diploid controls, the hippocampus from the Ts1Cje mouse had a smaller LTP and an increased LTD. The changes are less dramatic than had been reported previously for the Ts65Dn mouse. Furthermore, in the Ts1Cje mouse trains of pulses at both 20 Hz and 100 Hz produced a decrease in the evoked fEPSPs over the length of the train in comparison to diploid fEPSPs. These findings suggest that genes from Ts1Cje chromosome, including GIRK2 potassium channel, contribute to abnormal short- and long-term plasticity.
Assuntos
Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Plasticidade Neuronal/fisiologia , Trissomia/genética , Trissomia/fisiopatologia , Potenciais de Ação/genética , Animais , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Humanos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Camundongos , Camundongos Mutantes Neurológicos , Plasticidade Neuronal/efeitos dos fármacosRESUMO
Down syndrome (DS) results from the presence of an extra copy of genes on the long-arm of chromosome 21. Aberrant expression of these trisomic genes leads to widespread neurological changes that vary in their severity. However, how the presence of extra genes affects the physiological and behavioral phenotypes associated with DS is not well understood. The most likely cause of the complex DS phenotypes is the overexpression of dosage-sensitive genes. However, other factors, such as the complex interactions between gene products as proteins and noncoding RNAs, certainly play significant roles contributing to the spectrum of severity. Here we will review evidence regarding how the overexpression of one particular gene encoding for G-protein-activated inward rectifying potassium type 2 (GIRK2) channel subunit and its coupling to GABA(B) receptors may contribute to a range of mental and functional disabilities in DS.
Assuntos
Síndrome de Down/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptores de GABA-B/metabolismo , Transdução de Sinais , Animais , Transtornos Cognitivos/complicações , Transtornos Cognitivos/metabolismo , Síndrome de Down/complicações , Síndrome de Down/patologia , Hipocampo/metabolismo , Hipocampo/patologia , HumanosRESUMO
Down syndrome (DS) is the most common nonheritable cause of mental retardation. DS is the result of the presence of an extra chromosome 21 and its phenotype may be a consequence of overexpressed genes from that chromosome. One such gene is Kcnj6/Girk2, which encodes the G-protein-coupled inward rectifying potassium channel subunit 2 (GIRK2). We have recently shown that the DS mouse model, Ts65Dn, overexpresses GIRK2 throughout the brain and in particular the hippocampus. Here we report that this overexpression leads to a significant increase ( approximately 2-fold) in GABA(B)-mediated GIRK current in primary cultured hippocampal neurons. The dose response curves for peak and steady-state GIRK current density is significantly shifted left toward lower concentrations of baclofen in Ts65Dn neurons compared with diploid controls, consistent with increased functional expression of GIRK channels. Stationary fluctuation analysis of baclofen-induced GIRK current from Ts65Dn neurons indicated no significant change in single-channel conductance compared with diploid. However, significant increases in GIRK channel density was found in Ts65Dn neurons. In normalized baclofen-induced GIRK current and GIRK current kinetics no difference was found between diploid and Ts65Dn neurons, which suggests unimpaired mechanisms of interaction between GIRK channel and GABA(B) receptor. These results indicate that increased expression of GIRK2 containing channels have functional consequences that likely affect the balance between excitatory and inhibitory neuronal transmission.
Assuntos
Encéfalo/metabolismo , Síndrome de Down/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptores de GABA-B/metabolismo , Transmissão Sináptica/genética , Animais , Animais Recém-Nascidos , Baclofeno/farmacologia , Encéfalo/fisiopatologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Síndrome de Down/genética , Síndrome de Down/fisiopatologia , Potenciais Pós-Sinápticos Excitadores/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Agonistas GABAérgicos/farmacologia , Potenciais Pós-Sinápticos Inibidores/genética , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Receptores de GABA-B/genética , Trissomia/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
The Ts65Dn mouse model of Down syndrome (DS) has an extra segment of chromosome (Chr.) 16 exhibits abnormal behavior, synaptic plasticity and altered function of several signaling molecules. We have further investigated signaling pathways that may be responsible for the impaired hippocampal plasticity in the Ts65Dn mouse. Here we report that calcium/calmodulin-dependent protein kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase (ERK), protein kinase A (PKA) and protein kinase C (PKC), all of which have been shown to be involved in synaptic plasticity, are altered in the Ts65Dn hippocampus. We found that the phosphorylation of CaMKII and protein kinase Akt was increased, whereas ERK was decreased. Activities of PKA and PKC were decreased. Furthermore, abnormal PKC activity and an absence of the increase in Akt phosphorylation were demonstrated in the Ts65Dn hippocampus after high-frequency stimulation that induces long-term potentiation. Our findings suggest that abnormal synaptic plasticity in the Ts65Dn hippocampus is the result of compensatory alterations involving the glutamate receptor subunit GluR1 in either one or more of these signaling cascades caused by the expression of genes located on the extra segment of Chr. 16.
Assuntos
Síndrome de Down/fisiopatologia , Hipocampo/fisiologia , Plasticidade Neuronal/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Estimulação Elétrica , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteína Quinase C/fisiologia , Receptor Cross-Talk/fisiologia , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
1. Down syndrome (DS) arises from the presence of three copies of chromosome (Chr.) 21. Fine motor learning deficits found in DS from childhood to adulthood result from expression of extra genes on Chr. 21, however, it remains unclear which if any of these genes are the specific causes of the cognitive and motor dysfunction. DS cerebellum displays morphological abnormalities that likely contribute to the DS motor phenotype. 2. The G-protein-activated inwardly rectifying potassium channel subunit 2 (GIRK2) is expressed in cerebellum and can shunt dendritic conductance and attenuate postsynaptic potentials. We have used an interbreeding approach to cross a genetic mouse model of DS (Ts65Dn) with Girk2 knockout mice and examined its relative expression level by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. 3. We report here for the first time that GIRK2 is expressed in unipolar brush cells, which are excitatory interneurons of the vestibulocerebellum and dorsal cochlear nucleus. Analysis of disomic-Ts65Dn/Girk2((+/+/-)) and heterozygous-Diploid/Girk2((+/-)) mice shows that GIRK2 expression in Ts65Dn lobule X follows gene dosage. The lobule X of Ts65Dn mice contain greater numbers of unipolar brush cells co-expressing GIRK2 and calretinin than the control mouse groups. 4. These results demonstrate that gene triplication can impact specific cell types in the cerebellum. We hypothesize that GIRK2 overexpression will adversely affect cerebellar circuitry in Ts65Dn vestibulocerebellum and dorsal cochlear nucleus due to GIRK2 shunting properties and its effects on resting membrane potential.