Reduced cortical inhibition in a mouse model of familial childhood absence epilepsy

Mutations in the GABA(A) receptor gamma2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a gamma2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of gamma2(R43Q) was seen in heterozygous mice in the absence of any change in alpha1 subunit surface expression, ruling out a dominant-negative effect. GABA(A)-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.     

Clinical characteristics and causative genescreening in Chinese families with autosomal domi-nant lateral temporal lobe epilepsy

正Objective To explore the clinical,imaging,EEG characteristic and genetic cause of autosomal dominant lateral temporal lobe epilepsy (ADLTE) in Chinese ADLTE patients from four Han Chinese families. Methods The clinical data,electrophysiological findings and brain images of Chinese ADLTE patients from four Han     

Enhanced GABAergic inhibition preserves hippocampal structure and function in a model of epilepsy.

Extensive electrical stimulation of the perforant pathway input to the hippocampus results in a characteristic pattern of neuronal death, which is accompanied by an impairment of cognitive functions similar to that seen in human temporal lobe epilepsy. The excitotoxic hypothesis of epileptic cell death [Olney, J. W. (1978) in Kainic Acid as a Tool in Neurobiology, eds. McGeer, E., Olney, J. W. & McGeer, P. (Raven, New York), pp. 95-121; Olney, J. W. (1983) in Excitotoxins, eds. Fuxe, K., Roberts, P. J. & Schwartch, R. (Wenner-Gren International Symposium Series, Macmillan, London), Vol. 39, pp. 82-96; and Rothman, S. M. & Olney, J. W. (1986) Ann. Neurol. 19, 105-111] predicts an imbalance between excitation and inhibition, which occurs probably as a result of hyperactivity in afferent pathways or impaired inhibition. In the present study, we investigated whether the enhancement of gamma-aminobutyric acid (GABA)-mediated (GABAergic) inhibition of neurotransmission by blocking the GABA-metabolizing enzyme, GABA transaminase, could influence the histopathological and/or the behavioral outcome in this epilepsy model. We demonstrate that the loss of pyramidal cells and hilar somatostatin-containing neurons can be abolished by enhancing the level of synaptically released GABA, and that the preservation of hippocampal structure is accompanied by a significant sparing of spatial memory as compared with placebo-treated controls. These results suggest that enhanced GABAergic inhibition can effectively block the pathophysiological processes that lead to excitotoxic cell death and, as a result, protect the brain from seizure-induced cognitive impairment.     

Impaired hippocampal rhythmogenesis in a mouse model of mesial temporal lobe epilepsy

Mesial temporal lobe epilepsy (mTLE) is one of the most common forms of epilepsy, characterized by hippocampal sclerosis and memory deficits. Injection of kainic acid (KA) into the dorsal hippocampus of mice reproduces major electrophysiological and histopathological characteristics of mTLE. In extracellular recordings from the morphologically intact ventral hippocampus of KA-injected epileptic mice, we found that theta-frequency oscillations were abolished, whereas gamma oscillations persisted both in vivo and in vitro. Whole-cell recordings further showed that oriens-lacunosum-moleculare (O-LM) interneurons, key players in the generation of theta rhythm, displayed marked changes in their intrinsic and synaptic properties. Hyperpolarization-activated mixed cation currents (Ih) were significantly reduced, resulting in an increase in the input resistance and a hyperpolarizing shift in the resting membrane potential. Additionally, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) was increased, indicating a stronger excitatory input to these neurons. As a consequence, O-LM interneurons increased their firing rate from theta to gamma frequencies during induced network activity in acute slices from KA-injected mice. Thus, our physiological data together with network simulations suggest that changes in excitatory input and synaptic integration in O-LM interneurons lead to impaired rhythmogenesis in the hippocampus that in turn may underlie memory deficit.     

Role of proinflammatory cytokines in the inhibition of hippocampal neurogenesis in mesial temporal lobe epilepsy

Background

In mesial temporal lobe epilepsy, reduced hippocampal neurogenesis correlates with severe impairment in declarative learning and memory. The proinflammatory cytokines interleukin 1β and high-mobility group box 1 (HMGB1) and their receptors have key roles in generating and perpetuating seizures. This study aimed to assess their potential role in inhibiting neurogenesis in patients with mesial temporal lobe epilepsy and whether any effect would be pharmacologically reversible as a possible mechanism to restore learning and memory deficits.

Biochemical studies on frontal lobe white matter of young and old human brains

Frontal lobe white matter obtained at autopsy from 5 young (45–60 yr) and 5 non-demented old (77–78 yr) individuals was studied. Water, total protein, myelin basic protein (BP) content and 2′,3′-cyclic nucleotide 3′phosphohydrolase (CNP) activity in the frontal white matter of the two groups were compared. Myelin BP was measured by two different methods, RIA and batch extraction. Results indicated no significant changes in water and in total protein content. Myelin BP, as measured by both methods, however, was found to be significantly decreased in brains of the older group. CNP activity was also decreased but only slightly. This data suggests that with advancing age, myelin may undergo some qualitative changes.     

Immunolocalization of ion transport proteins in human autosomal dominant polycystic kidney epithelial cells.

The kidneys of patients with autosomal dominant polycystic kidney disease become massively enlarged due to the progressive expansion of myriad fluid-filled cysts. The epithelial cells that line the cyst walls are responsible for secreting the cyst fluid, but the mechanism through which this secretion occurs is not well established. Recent studies suggest that renal cyst epithelial cells actively secrete Cl across their apical membranes, which in turn drives the transepithelial movement of Na and water. The characteristics of this secretory flux suggest that it is dependent upon the participation of an apical cystic fibrosis transmembrane conductance regulator (CFTR)-like Cl channel and basolateral Na,K-ATPase. To test this hypothesis, we have immunolocalized the CFTR and Na,K-ATPase proteins in intact cysts and in cyst epithelial cells cultured in vitro on permeable filter supports. In both settings, cyst epithelial cells were found to possess Na,K-ATPase exclusively at their basolateral surfaces; apical labeling was not detected. The CFTR protein was present at the apical surfaces of cyst epithelial cells that had been stimulated to secrete through incubation in forskolin. CFTR was detected in intracellular structures in cultured cyst epithelial cells that had not received the forskolin treatment. These results demonstrate that the renal epithelial cells that line cysts in autosomal dominant polycystic kidney disease express transport systems with the appropriate polarity to mediate active Cl and fluid secretion.     

Inheritance of human breast cancer: evidence for autosomal dominant transmission in high-risk families.

Segregation analysis of breast cancer in families can provide the logical basis and the specific genetic models for mapping and identifying genes responsible for human breast cancer. Patterns of breast cancer occurrence in families were investigated by complex segregation analysis. In a sample of 1579 nuclear families ascertained through a population-based series of probands, an autosomal dominant model with a highly penetrant susceptibility allele fully explained disease clustering. From the maximum-likelihood Mendelian model, the frequency of the susceptibility allele was 0.0006 in the general population, and lifetime risk of breast cancer was 0.82 among susceptible women and 0.08 among women without the susceptibility allele. Inherited susceptibility affected only 4% of families in the sample: multiple cases of this relatively common disease occurred in other families by chance. The same genetic models, with higher gene frequency, explained disease clustering in an extended kindred at high risk of breast cancer. Evidence for a highly penetrant, autosomal dominant susceptibility allele for breast cancer in a high-risk family and the general population suggests that high-risk families can serve as models for understanding breast cancer in the population as a whole.     

Hepatic and renal manifestations in autosomal dominant polycystic kidney disease: a dichotomy of two ends of a spectrum

Autosomal dominant polycystic kidney disease (ADPKD) is a multisystem disorder. It is the most common genetic cause of end-stage renal disease. One frequent extra-renal manifestation is hepatic cyst formation. The majority of ADPKD patients develop complications as a result of renal cyst formation; however, a small proportion develop extensive hepatic disease with minor renal features. Both phenotypes seem to represent the spectrum of ADPKD. This review discusses the current understanding of the pathogenesis of the disease, its manifestations and the mechanisms of cyst formation. Furthermore, it focuses on monitoring the disease and the treatment options currently available.     

Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a progressive genetic syndrome with an incidence of 1:500 in the population, arising from inherited mutations in the genes for polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2). Typical onset is in middle age, with gradual replacement of renal tissue with thousands of fluid-filled cysts, resulting in end-stage renal disease requiring dialysis or kidney transplantation. There currently are no approved therapies to slow or cure ADPKD. Mutations in the PKD1 and PKD2 genes abnormally activate multiple signaling proteins and pathways regulating cell proliferation, many of which we observe, through network construction, to be regulated by heat shock protein 90 (HSP90). Inhibiting HSP90 with a small molecule, STA-2842, induces the degradation of many ADPKD-relevant HSP90 client proteins in Pkd1^−/− primary kidney cells and in vivo. Using a conditional Cre-mediated mouse model to inactivate Pkd1 in vivo, we find that weekly administration of STA-2842 over 10 wk significantly reduces initial formation of renal cysts and kidney growth and slows the progression of these phenotypes in mice with preexisting cysts. These improved disease phenotypes are accompanied by improved indicators of kidney function and reduced expression and activity of HSP90 clients and their effectors, with the degree of inhibition correlating with cystic expansion in individual animals. Pharmacokinetic analysis indicates that HSP90 is overexpressed and HSP90 inhibitors are selectively retained in cystic versus normal kidney tissue, analogous to the situation observed in solid tumors. These results provide an initial justification for evaluating HSP90 inhibitors as therapeutic agents for ADPKD.     

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1^V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1^+/+ and Gpsm1^+/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.     

Simulation of human autosomal dominant retinitis pigmentosa in transgenic mice expressing a mutated murine opsin gene.

Autosomal dominant retinitis pigmentosa (ADRP), slowly progressing over decades, leads to severe visual impairment and in some cases to complete blindness. More than 40 mutations in the human opsin gene have been linked to some forms of this genetically heterogeneous disease. In photoreceptor cells of ADRP patients with mutations in the opsin gene, normal rhodopsin is thought to be synthesized concomitantly with mutated rhodopsin, which, by an unknown mechanism, causes the slow degeneration of the photoreceptor cells. To establish a transgenic mouse line that carries a mutated mouse opsin gene in addition to the endogenous opsin gene, we introduced a mouse opsin gene containing mutations in exon 1 into the germ line of a normal mouse. The alterations consisted of three amino acid substitutions near the N terminus of rhodopsin, Val-20-->Gly (V20G), Pro-23-->His (P23H), and Pro-27-->Leu (P27L). The P23H mutation is the most prevalent mutation in human ADRP patients. During early postnatal development, mice heterozygous for the mutated opsin gene appear to develop normal photoreceptors, but their light-sensitive outer segments never reach normal length. With advancing age, both rod and cone photoreceptors are reduced progressively in number. The slow degeneration of the transgenic retina is associated with a gradual decrease of light-evoked electroretinogram responses. Our results show that simultaneous expression of mutated and normal opsin genes induces a slow degeneration of both rod and cone photoreceptors and that the course of the retinal degeneration of the mutant mouse retina mimics the course of human ADRP.     

Arrhythmia phenotype in mouse models of human long QT

Enhanced dispersion of repolarization (DR) was proposed as a unifying mechanism, central to arrhythmia genesis in the long QT (LQT) syndrome. In mammalian hearts, K⁺ channels are heterogeneously expressed across the ventricles resulting in ‘intrinsic’ DR that may worsen in long QT. DR was shown to be central to the arrhythmia phenotype of transgenic mice with LQT caused by loss of function of the dominant mouse K⁺ currents. Here, we investigated the arrhythmia phenotype of mice with targeted deletions of KCNE1 and KCNH2 genes which encode for minK/IsK and Merg1 (mouse homolog of human ERG) proteins resulting in loss of function of I_Ks and I_Kr, respectively. Both currents are important human K⁺ currents associated with LQT5 and LQT2. Loss of minK, a protein subunit that interacts with KvLQT1, results in a marked reduction of I_Ks giving rise to the Jervell and Lange–Nielsen syndrome and the reduced KCNH2 gene reduces MERG and I_Kr. Hearts were perfused, stained with di-4-ANEPPS and optically mapped to compare action potential durations (APDs) and arrhythmia phenotype in homozygous minK (minK^−/−) and heterozygous Merg1 (Merg^+/−) deletions and littermate control mice. MinK^−/− mice has similar APDs and no arrhythmias (n = 4). Merg^+/− mice had prolonged APDs (from 20 ± 6 to 32 ± 9 ms at the base, p < 0.01; from 18 ± 5 to 25 ± 9 ms at the apex, p < 0.01; n = 8), longer refractory periods (RP) (36 ± 14 to 63 ± 27 at the base, p < 0.01 and 34 ± 5 to 53 ± 21 ms at the apex, p < 0.03; n = 8), higher DR 10.4 ± 4.1 vs. 14 ± 2.3 ms, p < 0.02) and similar conduction velocities (n = 8). Programmed stimulation exposed a higher propensity to VT in Merg^+/− mice (60% vs. 10%). A comparison of mouse models of LQT based on K⁺ channel mutations important to human and mouse repolarization emphasizes DR as a major determinant of arrhythmia vulnerability.     

Deficiency of rds/peripherin causes photoreceptor death in mouse models of digenic and dominant retinitis pigmentosa

Retinitis pigmentosa (RP) is a group of inherited blinding diseases caused by mutations in multiple genes including RDS. RDS encodes rds/peripherin (rds), a 36-kDa glycoprotein in the rims of rod and cone outer-segment (OS) discs. Rom1 is related to rds with similar membrane topology and the identical distribution in OS. In contrast to RDS, no mutations in ROM1 alone have been associated with retinal disease. However, an unusual digenic form of RP has been described. Affected individuals in several families were doubly heterozygous for a mutation in RDS causing a leucine 185 to proline substitution in rds (L185P) and a null mutation in ROM1. Neither mutation alone caused clinical abnormalities. Here, we generated transgenic/knockout mice that duplicate the amino acid substitutions and predicted levels of rds and rom1 in patients with RDS-mediated digenic and dominant RP. Photoreceptor degeneration in the mouse model of digenic RP was faster than in the wild-type and monogenic controls by histological, electroretinographic, and biochemical analysis. We observed a positive correlation between the rate of photoreceptor loss and the extent of OS disorganization in mice of several genotypes. Photoreceptor degeneration in RDS-mediated RP appears to be caused by a simple deficiency of rds and rom1. The critical threshold for the combined abundance of rds and rom1 is approximately 60% of wild type. Below this value, the extent of OS disorganization results in clinically significant photoreceptor degeneration.     

Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer

Antigen specific T cell migration to sites of infection or cancer is critical for an effective immune response. In mouse models of cancer, the number of lymphocytes reaching the tumor is typically only a few hundred, yet technology capable of imaging these cells using bioluminescence has yet to be achieved. A combination of codon optimization, removal of cryptic splice sites and retroviral modification was used to engineer an enhanced firefly luciferase (ffLuc) vector. Compared with ffLuc, T cells expressing our construct generated >100 times more light, permitting detection of as few as three cells implanted s.c. while maintaining long term coexpression of a reporter gene (Thy1.1). Expression of enhanced ffLuc in mouse T cells permitted the tracking of <3 × 10⁴ adoptively transferred T cells infiltrating sites of vaccination and preestablished tumors. Penetration of light through deep tissues, including the liver and spleen, was also observed. Finally, we were able to enumerate infiltrating mouse lymphocytes constituting <0.3% of total tumor cellularity, representing a significant improvement over standard methods of quantitation including flow cytometry.     

High levels of brain-type creatine kinase activity in human platelets and leukocytes: a genetic anomaly with autosomal dominant inheritance

The ectopic expression in peripheral blood cells of the brain-type creatine kinase (CKB) is an autosomal dominant inherited anomaly named CKBE (MIM ID 123270). Here, we characterized the CK activity in serum, platelets (PLT) and leukocytes (WBC) of 22 probands (from 8 unrelated families) and 10 controls. CK activity was measured by standard UV-photometry. Expression of the CKB gene was analyzed by real-time PCR and Western blotting. DNA sequencing including bisulfite treatment was used for molecular analysis of the CKB gene. Serum CK levels were comparable between probands and controls. CKBE probands revealed significantly higher CK activity in PLT (3.7 ± 2.7 versus 179.2 ± 83.0 U/10(12) PLT; p<0.001) and WBC (0.4 ± 0.3 versus 2.6 ± 2.1 U/10(9) WBC; p=0.004). Inhibitory anti-CKM antibodies did not affect CK activity indicating that the CK activity is generated exclusively by the CK-BB isoenzyme. CKB mRNA and protein levels were significantly higher in PLT and WBC from probands compared to controls. Re-sequencing of the entire CKB gene and methylation analysis of a CpG island revealed no alteration in CKBE probands. The genetic basis of CKBE remains unclear, however, we propose that a de-methylated CKB gene is inherited that leads to high CKB expression levels in myeloic precursor cells in the bone marrow.     

Evidence for genetic heterogeneity of pseudohypoaldosteronism type 1: identification of a novel mutation in the human mineralocorticoid receptor in one sporadic case and no mutations in two autosomal dominant kindreds

Pseudohypoaldosteronism type 1 (PHA1) is characterized by neonatal salt wasting resistant to mineralocorticoids. There are 2 forms of PHA1: the autosomal recessive form with symptoms persisting into adulthood, caused by mutations in the amiloride-sensitive luminal sodium channel, and the autosomal dominant or sporadic form, which shows milder symptoms that remit with age. Mutations in the gene encoding the human mineralocorticoid receptor (hMR) are, at least in some patients, responsible for the latter form of PHA1. We here report the results of a genetic study in a sporadic case and in 5 affected patients from 2 families with autosomal dominant PHA1. In the sporadic case we identified a new frameshift mutation, Ins2871C, in exon 9 of the hMR gene. Family members were asymptomatic and had no mutation. This mutation is the first described in exon 9 and impairs the last 27 amino acids of the hormone-binding domain. In 2 kindreds with autosomal dominant PHA1 we found no mutation of the hMR gene. Our results confirm the hypothesis that autosomal dominant or sporadic PHA1 is a genetically heterogeneous disease involving other, as yet unidentified, genes.     

人鼠嵌合肝小鼠模型的建立及在HBV致病研究中的应用

病毒性肝炎一直是全球范围内一个沉重的肝病负担,其中以乙型肝炎最为严重。由于HBV仅感染灵长类动物,因此其所致肝病很难在动物模型中被复制。人鼠嵌合肝小鼠模型是指将人源肝细胞移植入免疫缺陷的肝损伤小鼠肝内而形成的嵌合体小鼠。由于小鼠肝内嵌合有人肝细胞,可以在其体内模拟HBV的感染,利于进行进一步的机制研究。目前该模型主要以白蛋白启动子调控尿激酶(Alb-uPA)小鼠模型和延胡索酰乙酰乙酸水解酶(Fah~(-/-))小鼠模型为基础,在人源肝组织和外周血中均可检测到HBV,并已应用于HBV突变、HBV基因功能、抗病毒药物效果以及HBV与免疫系统的相互作用等方面的研究。本文就传统HBV感染动物模型的利弊、人鼠嵌合肝小鼠模型的研究进展和改进方法进行综述。     

Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene.

A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.