A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1(tm1Apa) mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1(tm1Apa) homozygous mice display deficits in dendritic delivery of Stau1-EYFP and beta-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1(tm1Apa) mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function.     

AMPA receptor and GEF-H1/Lfc complex regulates dendritic spine development through RhoA signaling cascade

AMPA receptors (AMPA-R) are major mediators of synaptic transmission and plasticity in the developing and adult central nervous system. Activity-dependent structural plasticity mediated by dynamic changes in the morphology of spines and dendrites is also essential for the formation and tuning of neuronal circuits. RhoA and Rac1 are known to play important roles in the regulation of spine and dendrite development in response to neuronal activity. These Rho GTPases are activated by guanine nucleotide exchange factors (GEFs). In this study, we identified GEF-H1/Lfc as a component of the AMPA-R complex in the brain. GEF-H1 is enriched in the postsynaptic density and is colocalized with GluR1 at spines. GEF-H1 activity negatively regulates spine density and length through a RhoA signaling cascade. In addition, AMPA-R-dependent changes in spine development are eliminated by down-regulation of GEF-H1. Altogether, these results strongly suggest that GEF-H1 is an important mediator of AMPA-R activity-dependent structural plasticity in neurons.     

Targeted dendrotomy reveals active and passive contributions of the dendritic tree to synaptic integration and neuronal output

Neurons typically function as transduction devices, converting patterns of synaptic inputs, received on the dendrites, into trains of output action potentials in the axon. This transduction process is surprisingly complex and has been proposed to involve a two-way dialogue between axosomatic and dendritic compartments that can generate mutually interacting regenerative responses. To manipulate this process, we have developed a new approach for rapid and reversible occlusion or amputation of the primary dendrites of individual neurons in brain slices. By applying these techniques to cerebellar Purkinje and layer 5 cortical pyramidal neurons, we show directly that both the active and passive properties of dendrites differentially affect firing in the axon depending on the strength of stimulation. For weak excitation, dendrites act as a passive electrical load, raising spike threshold and dampening axonal excitability. For strong excitation, dendrites contribute regenerative inward currents, which trigger burst firing and enhance neuronal excitability. These findings provide direct support for the idea that dendritic morphology and conductances act in concert to regulate the excitability of the neuron.     

SCN5A基因移码突变导致Brugada综合征

目的：检测Brugada综合征的致病基因突变位点。方法：对1个Brugada综合征家系11名成员和20名正常人的DNA样本应用双脱氧链终止基因测序法进行心脏钠离子通道α亚单位（voltage-gated sodium channel type V,SCN5A)基因测序。结果：SCN5A基因测序发现Brugada综合征家系第22个外显子存在1个杂合基因移码突变位点，经克隆传代后测序发现该突变为4087insC。该突变使通道蛋白1314－1317位氨基酸发生改变并在1318位终止，导致钠离子通道第3结构域S4结构变化，S5－6及第4结构域全部缺失。该突变在Brugada综合征家系中分布符合常染色体显性分布规律。对照组未发现相同突变。结论：SCN5A基因4087insC是国内首次发现的导致Brugada 综合征的基因突变位点，也是国际上发现的第2个引起Brugada综合征的SCN5A基因移码突变     

Autosomal dominant transmission of diabetes and congenital hearing impairment secondary to a missense mutation in the WFS1 gene

Aims Mutations of the WFS1 gene have been implicated in autosomal dominant diseases, such as low‐frequency sensorineural hearing impairment (LFSNHI) and/or diabetes mellitus and/or optic atrophy. The aim was to investigate WFS1 gene sequences in a family with diabetes mellitus and hearing impairment. Methods Three members of a family with a maternally inherited combination of diabetes mellitus and hearing impairment, but no specific mutations in its mitochondrial genome, were investigated for mutations in the WFS1 gene. Results This pedigree, in which the proband had non‐insulin‐dependent diabetes mellitus and congenital hearing impairment and his mother a triple combination of diabetes mellitus, hearing impairment and optic atrophy, was found to be associated with autosomal dominant transmission of the E864K mutation of the WFS1 gene. Conclusions In the light of this confirmatory study, we recommend the systematic analysis of WFS1 gene sequences in patients with parentally inherited diabetes mellitus and deafness (± optic atrophy), in particular when diabetogenic mtDNA mutations have been excluded.     

A novel N491S mutation in the human SLC11A2 gene impairs protein trafficking and in association with the G212V mutation leads to microcytic anemia and liver iron overload

Background

DMT1 is a transmembrane iron transporter involved in iron duodenal absorption and cellular iron uptake. Mutations in the human SLC11A2 gene coding DMT1 lead to microcytic anemia and hepatic iron overload, with unexpectedly low levels of plasma ferritin in the presence of iron stores.

Design and methods

We report a patient with a similar phenotype due to two mutations in the SLC11A2 gene, the known p.Gly212Val (G212V) mutation and a novel one, p.Asn491Ser (N491S). To assess the expression of DMT1 in human liver, we studied the expression of the four DMT1 mRNA isoforms by real-time quantitative PCR in control human liver samples. We also studied the effect of G212V and N491S DMT1 mutations on RNA splicing in blood leukocytes and cellular trafficking of dsRed2-tagged-DMT1 protein in the human hepatic cell line HuH7.

Results

Our results showed that i) only the isoforms 1B-IRE and 1B-nonIRE were significantly expressed in human liver; ii) the G212V mutation did not seem to affect mRNA splicing and the N491S mutation induced a splicing alteration leading to a truncated protein, which seemed quantitatively of low relevance; and iii) the N491S mutation, in contrast to the G212V mutation, led to abnormal protein trafficking.

Conclusions

Our data confirm the major role of DMT1 in the maintenance of iron homeostasis in humans and demonstrate that the N491S mutation, through its deleterious effect on protein trafficking, contributes together with the G212V mutation to the development of anemia and hepatic iron overload.     

Selective delivery of beta cell antigen to dendritic cells in vivo leads to deletion and tolerance of autoreactive CD8+ T cells in NOD mice

Type 1 diabetes (T1D) is an autoimmune disease resulting from defects in central and peripheral tolerance and characterized by T cell-mediated destruction of islet beta cells. Cytotoxic CD8(+) T cells, reactive to beta cell antigens, are required for T1D development in the NOD mouse model of the disease, and CD8(+) T cells specific for beta cell antigens can be detected in the peripheral blood of T1D patients. It has been evident that in nonautoimmune-prone mice, dendritic cells (DCs) present model antigens in a tolerogenic manner in the steady state, e.g., in the absence of infection, and cause T cells to proliferate initially but then to be deleted or rendered unresponsive. However, this fundamental concept has not been evaluated in the setting of a spontaneous autoimmune disease. To do so, we delivered a mimotope peptide, recognized by the diabetogenic CD8(+) T cell clone AI4, to DCs in NOD mice via the endocytic receptor DEC-205. Proliferation of transferred antigen-specific T cells was initially observed, but this was followed by deletion. Tolerance was achieved because rechallenge of mice with the mimotope peptide in adjuvant did not induce an immune response. Thus, targeting of DCs with beta cell antigens leads to deletion of autoreactive CD8(+) T cells even in the context of ongoing autoimmunity in NOD mice with known tolerance defects. Our results provide support for the development of DC targeting of self antigens for treatment of chronic T cell-mediated autoimmune diseases.     

A missense mutation in the human melanocortin-4 receptor gene in relation to abdominal obesity and salivary cortisol

Aims/hypothesis: The melanocortin-4 receptor (MC4-R) regulates food intake and possibly energy expenditure, and the inactivation of the MC4-R by gene targeting results in obesity, a phenotype strongly associated with Type II (non-insulin-dependent) diabetes mellitus. In our study, we addressed the hypothesis that a G?A substitution at codon 103 (Val-103Ile) of the MC4R gene influences abdominal obesity, insulin, glucose, and lipid metabolism as well as circulating hormones, including salivary cortisol. Methods: We genotyped the missense variant at codon 103 of the MC4R gene in 284 unrelated Swedish men born in 1944 by using polymerase chain reaction amplification followed by digestion with the restriction enzyme HincII. Results: The frequency of allele G was 0.97 and 0.03 for allele A. The observed genotype frequencies were 95 % and 5 % for G/G and G/A, respectively. The heterozygotes had lower waist-to-hip ratio (p = 0.023) and trends for lower body mass index (p = 0.054) and abdominal sagittal diameter (p = 0.095) compared to G/G homozygotes. Moreover, G/A subjects had borderline lower serum leptin concentrations (p = 0.087) and total cholesterol (p = 0.082). The heterozygotes had also, in comparison to G/G subjects, significantly (p < 0.01) higher mean cortisol concentrations in the morning (21.4 vs 14.6 nmol/l), at ll:45 h (11.6 vs 7.0 nmol/l), 30 min after a standardized lunch (15.3 vs 8.0 nmol/l) and finally, 60 min after lunch (10.8 vs 6.7 nmol/l). Conclusion/interpretation: These findings suggest that the missense mutation in the MC4R gene could contribute to the variability in body mass, abdominal fat distribution and leptin concentrations in the general population. Moreover, the G/A mutation exhibits evidence of associations with diurnal cortisol levels. [Diabetologia (2001) 44: 1335–1338] Received: 11 April 2001 and in revised form: 8 June 2001     

A missense mutation in the hepatocyte nuclear factor 4 alpha gene in a UK pedigree with maturity-onset diabetes of the young

Summary Maturity-onset diabetes of the young (MODY) is a monogenic subgroup of non-insulin dependent diabetes mellitus (NIDDM) characterised by an early age of onset (< 25 years) and an autosomal dominant mode of inheritance. MODY is genetically heterogeneous with three different genes identified to date; hepatocyte nuclear factor 4 alpha (HNF-4α) [MODY1], glucokinase [MODY2] and hepatocyte nuclear factor 1 alpha (HNF-1α) [MODY3]. A nonsense mutation in the HNF-4α gene has recently been shown to cause MODY in a single large North American pedigree (RW). We screened a large UK Caucasian MODY family which showed weak evidence of linkage to the MODY1 locus on chromosome 20q (lod score for ADA 0.68 at θ = 0) for mutations in the coding region of the HNF-4α gene by direct sequencing. A missense mutation resulting in the substitution of glutamine for glutamic acid was identified in exon 7 (E276Q). The mutation was present in all of the diabetic members of the pedigree plus two unaffected subjects and was not detected in 75 normal control subjects or 95 UK Caucasian subjects with late-onset NIDDM. This is the first missense mutation to be described in the HNF-4α gene. [Diabetologia (1997) 40: 859–862] Received: 7 March 1997 and in revised form: 16 April 1997     

Self‐reported history of overweight and its relationship to disordered eating in adolescent girls with Type 1 diabetes

Aims Increased body weight and disordered eating attitudes/behaviours are common in adolescent girls with Type 1 diabetes (T1D). Disordered eating increases risks for diabetes‐related complications. This study aimed to identify a rapid screening approach for disordered eating attitudes and behaviours in adolescent girls with T1D and to examine the relationship between disordered eating and body weight in this population. Methods Ninety adolescent girls, aged 12–19 years, provided a self‐assessment of weight status. Participants also completed questionnaires to assess attitudes/behaviours toward food and eating, appetitive responsiveness to the food environment, disinhibition in eating and weight history. Results Forty‐three per cent of participants reported a history of overweight. Compared with participants who reported never being overweight, those who reported ever being overweight were significantly older, scored significantly higher on all measures of disordered eating attitudes/behaviours (P ≤ 0.009) and were 4.8 times more likely to be currently overweight or obese (P < 0.001). Glycated haemoglobin (HbA_1c) was similar between those who did and did not report ever being overweight. Conclusions Because of the ill‐health effects of disordered eating and the higher rate of overweight in adolescent girls with T1D, effective screening tools are warranted. The single question ‘Have you ever been overweight?’ may be sufficient as a first question to screen for those at high risk for disordered eating attitudes/behaviours and to provide early intervention and prevention.     

Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas

Although inappropriate activation of the Wnt/beta-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, we constructed a mutant mouse strain, Apc(lox/lox), in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity [10(9) plaque-forming units (pfu) per mouse] inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. beta-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of beta-catenin in the hepatocytes and by the induction of beta-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, we inoculated mice with lower doses of AdCre (0.5 x 10(9) pfu per mouse), compatible with both survival and persistence of beta-catenin-activated cells. In these conditions, 67% of mice developed HCC. beta-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/beta-catenin pathway by invalidation of Apc is required for liver tumorigenesis.