Anomalous functional organization of barrel cortex in GAP-43 deficient mice

Growth associated protein 43 (GAP-43), found only in the nervous system, regulates the response of neurons to axon guidance signals. It is also critical for establishing normal somatotopy. Mice lacking GAP-43 (KO) show aberrant pathfinding by thalamocortical afferents, and do not form cortical whisker/barrels. GAP-43 heterozygous (HZ) mice show more subtle deficits--delayed barrel segregation and enlarged barrels at postnatal day 7. Here, we used cortical intrinsic signal imaging to characterize adult somatotopy in wildtype (WT), GAP-43 KO, and HZ mice. We found clear foci of activation in GAP-43 KO cortex in response to single-whisker stimulation. However, the KO spatial activation patterns showed severe anomalies, indicating a loss of functional somatotopy. In some cases, multiple foci were activated by single whiskers, while in other cases, the same cortical zone was activated by several whiskers. The results are consistent with our previous findings of aberrant pathfinding and clustering by thalamocortical afferent axons, and absence of barrel patterning. Our findings indicate that cortex acts to cluster afferents from a given whisker, even in the absence of normal topography. By contrast, single-whisker stimulation revealed normal adult topographic organization in WT and HZ mice. However, we found that functional representations of adult HZ barrels are larger than those found in WT mice. Since histological HZ barrels recover normal dimensions by postnatal day 26, the altered circuit function in GAP-43 HZ cortex could be a secondary consequence of the rescue of barrel dimensions.     

Gene transfer of connexin43 into skeletal muscle

Cellular cardiomyoplasty using skeletal myoblasts may be beneficial for infarct repair. One drawback to skeletal muscle cells is their lack of gap junction expression after differentiation, thus preventing electrical coupling to host cardiomyocytes. We sought to overexpress the gap junction protein connexin43 (Cx43) in differentiated skeletal myotubes, using retroviral, adenoviral, and plasmid-mediated gene transfer. All strategies resulted in overexpression of Cx43 in cultured myotubes, but expression of Cx43 from constitutive viral promoters caused significant death upon differentiation. Dye transfer studies showed that surviving myotubes contained functional gap junctions, however. Retrovirally transfected myoblasts did not express Cx43 after grafting into the heart, possibly due to promoter silencing. Adenovirally transfected myoblasts expressed abundant Cx43 after forming myotubes in cardiac grafts, but grafts showed signs of injury at 1 week and had died by 2 weeks. Interestingly, transfection of already differentiated myotubes with adenoviral Cx43 was nontoxic, implying a window of vulnerability during differentiation. To test this hypothesis, Cx43 was expressed from the muscle creatine kinase (MCK) promoter, which is active only after myocyte differentiation. The MCK promoter resulted in high levels of Cx43 expression in differentiated myotubes but did not cause cell death during differentiation. MCK-Cx43-transfected myoblasts formed viable cardiac grafts and, in some cases, Cx43-expressing myotubes were in close apposition to host cardiomyocytes, possibly allowing electrical coupling. Thus, high levels of Cx43 during skeletal muscle differentiation cause cell death. When, however, expression of Cx43 is delayed until after differentiation, using the MCK promoter, myotubes are viable and express gap junction proteins after grafting in the heart. This strategy may permit electrical coupling of skeletal and cardiac muscle for cardiac repair.     

心室纤颤时心肌缝隙连接蛋白Cx43的变化

目的 观察心室纤颤(室颤)发生后缝隙连接蛋白Cx43的表达以及缝隙连接改造剂ZP123对Cx43表达的影响.方法 按照随机数字表法将30只家猪分为假手术组、模型组和ZP123干预组,每组10只.以80 V电压持续刺激动物5 s诱发室颤;致颤前15 min ZP123组给予ZP123 1μg/kg静脉推注+ZP12310μg·kg-1·h-1微量泵泵入;模型组泵入生理盐水50 ml;假手术组动物不致颤也不补液.室颤持续8 min后开胸取左心室游离壁心肌,用免疫荧光结合激光共聚焦显微镜技术检测Cx43的分布及水平,用蛋白质免疫印迹法(Western blotting)定量检测Cx43蛋白表达.结果 假手术组Cx43荧光信号强,分布均匀;模型组Cx43荧光信号弱,呈不均一分布;ZP123干预组Cx43荧光信号增强,不均一分布减轻.与假手术组比较,模型组心室肌组织Cx43荧光信号面积百分比、积分吸光度(A)值及蛋白表达均明显下降[面积百分比:(0.64±0.36)%比(1.27±0.19)%,积分A值:15 201±2 613比30 634±4 975,Cx43蛋白表达:0.72±0.08比0.97±0.07,均P＜0.05];与模型组比较,ZP123干预组Cx43表达[面积百分比(0.96±0.16)%,积分A值22 100±4 404,Cx43蛋白表达0.82±0.04]均明显升高(均P＜0.05).结论 室颤发生时心肌组织Cx43表达减少;应用ZP123可减少或逆转Cx43的降解.     

Connexin43-dependent mechanism modulates renin secretion and hypertension

To investigate the function of Cx43 during hypertension, we studied the mouse line Cx43KI32 (KI32), in which the coding region of Cx32 replaces that of Cx43. Within the kidneys of homozygous KI32 mice, Cx32 was expressed in cortical and medullary tubules, as well as in some extra- and intraglomerular vessels, i.e., at sites where Cx32 and Cx43 are found in WT mice. Under such conditions, renin expression was much reduced compared with that observed in the kidneys of WT and heterozygous KI32 littermates. After exposure to a high-salt diet, all mice retained a normal blood pressure. However, whereas the levels of renin were significantly reduced in the kidneys of WT and heterozygous KI32 mice, reaching levels comparable to those observed in homozygous littermates, they were not further affected in the latter animals. Four weeks after the clipping of a renal artery (the 2-kidney, 1-clip [2K1C] model), 2K1C WT and heterozygous mice showed an increase in blood pressure and in the circulating levels of renin, whereas 2K1C homozygous littermates remained normotensive and showed unchanged plasma renin activity. Hypertensive, but not normotensive, mice also developed cardiac hypertrophy. The data indicate that replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin, thus preventing the renin-dependent hypertension that is normally induced in the 2K1C model.     

Slow ventricular conduction in mice heterozygous for a connexin43 null mutation.

To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.     

Regulation of hematopoietic stem cell trafficking and mobilization by the endocannabinoid system

The cannabinoid receptors CB(1) and CB(2) are seven-transmembrane Gαi protein-coupled receptors and are expressed in certain mature hematopoietic cells. We recently showed that these receptors are expressed in murine and human hematopoietic stem cells (HSCs) and that CB(2) agonists induced chemotaxis, enhanced colony formation of marrow cells, as well as caused in vivo mobilization of murine HSCs with short- and long-term repopulating abilities. Based on these observations, we have further explored the role of CB(2) and its agonist AM1241 on hematopoietic recovery following sublethal irradiation in mice. Cannabinoid receptor 2 knockout mice (Cnr2(-/-) deficient mice) exhibited impaired recovery following sublethal irradiation as compared with irradiated wild-type (WT) mice, as determined by low colony-forming units and low peripheral blood counts. WT mice treated with CB(2) agonist AM1241 following sublethal irradiation demonstrated accelerated marrow recovery and increased total marrow cells (approximately twofold) and total lineage- c-kit(+) cells (approximately sevenfold) as well as enhanced HSC survival as compared with vehicle control-treated mice. When the CB(2) agonist AM1241 was administered to WT mice 12 days before their sublethal irradiation, analysis of hematopoiesis in these mice showed decreased apoptosis of HSCs, enhanced survival of HSCs, as well as increase in total marrow cells and c-kit+ cells in the marrow. Thus, CB(2) agonist AM1241 promoted recovery after sublethal irradiation by inhibiting apoptosis of HSCs and promoting survival, as well as enhancing the number of HSCs entering the cell cycle.     

室性心动过速时连接蛋白43含量与分布的变化

目的　探讨室性心动过速 (室速 )时心肌细胞连接蛋白 4 3(Cx 4 3)含量和分布的变化。方法　实验用日本大耳白兔 2 0只 ,随机分为对照组、30min室速组、6 0min室速组、12 0min室速组 4组。分别通过心室刺激复制室性心动过速动物模型 ,应用激光共聚焦显微镜技术和荧光免疫组织化学方法对其发生心律失常心肌连接蛋白 4 3的含量和分布进行定量分析。结果　 30min室速组连接蛋白 4 3像素密度较对照组减少 18.4 % (P <0 .0 5 ) ,6 0min室速组减少 38.0 % (P <0 .0 1) ,12 0min室速组减少 5 4 .8% (P <0 .0 1)。对照组各层间心肌细胞连接蛋白 4 3含量比较无显著差异 ,但心律失常后 ,中间层心肌细胞连接蛋白 4 3含量较其他层心肌细胞含量下降更明显 (P <0 .0 5或P <0 .0 1)。结论　室性心动过速时连接蛋白 4 3迅速降解 ,其分布也发生明显的改变 ,各层心肌细胞连接蛋白 4 3的降解程度也明显不均一。     

Fucose‐deficient hematopoietic stem cells have decreased self‐renewal and aberrant marrow niche occupancy

BACKGROUND: Modification of Notch receptors by O‐linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self‐renewal and stem cell niche features of fucose‐deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX^?/? mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two‐photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose‐deficient and wild‐type (WT) HSCs. RESULTS: Marrow progenitors from FX^?/? mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose‐deficient HSCs from FX^?/? mice displayed decreased self‐renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two‐photon intravital imaging, the fucose‐deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX^?/? mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O‐fucosylation, suggests that fucosylation‐modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.     

缝隙连接蛋白Cx32与Cx43在癫痫发病中的作用

目的:探讨缝隙连接蛋白(Cx)与癫痫之间的关系.方法:采用免疫组织化学方法测定氯化锂-匹罗卡品致痫大鼠癫痫发作后不同时间不同脑区Cx32与Cx43免疫阳性表达情况.结果:与对照组比较,致痫组大鼠海马区与皮层区Cx32和Cx43免疫阳性表达在癫痫发作1h后开始增强(Cx32免疫阳性细胞数分别为海马区16.62±4.51和皮层区14.85±3.30,均P＜0.05;Cx43免疫阳性细胞数分别为海马区18.26±4.03和皮层区18.65±4.51,均P＜0.01),24 h达到高峰(Cx32免疫阳性细胞数分别为海马区46.53±9.47和皮层区28.25±8.69,均P＜0.01;Cx43免疫阳性细胞数分别为海马区39.77±7.79和皮层区26.50±6.56,均P＜0.01),此后逐步下降.但至14 d Cx32海马区与皮层区免疫阳性细胞数分别为22.45±6.56和15.92±3.16,仍高于对照组(均P＜0.05),而Cx43在14 d的表达则有所不同:海马区免疫阳性细胞数17.54±3.77仍高于对照组(P＜0.01);皮层区表达则降至正常水平.致痫24 h时海马区Cx32和Cx43免疫阳性表达均明显高于同一时限的皮层区(均P＜0.05).结论:Cx32和Cx43参与了癫痫的发生与发展过程,反映了脑组织中神经元、星形胶质细胞之间的缝隙连接与癫痫发病机制密切相关.     

Lnk controls mouse hematopoietic stem cell self-renewal and quiescence through direct interactions with JAK2

In addition to its role in megakaryocyte production, signaling initiated by thrombopoietin (TPO) activation of its receptor, myeloproliferative leukemia virus protooncogene (c-Mpl, or Mpl), controls HSC homeostasis and self-renewal. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. We found that HSCs from Lnk-/- mice have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeat treatments with cytoablative 5-fluorouracil in vivo compared with WT HSCs. We further provide genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal, predominantly through Mpl. Consistent with this observation, Lnk-/- HSCs displayed potentiated activation of JAK2 specifically in response to TPO. Biochemical experiments revealed that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Of note, the JAK2 V617F mutant, found at high frequencies in myeloproliferative diseases, retains the ability to bind Lnk. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells and TPO/Mpl/JAK2/Lnk as a major regulatory pathway in controlling stem cell self-renewal and quiescence.     

Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.     

Correction of metachromatic leukodystrophy in the mouse model by transplantation of genetically modified hematopoietic stem cells

Gene-based delivery can establish a sustained supply of therapeutic proteins within the nervous system. For diseases characterized by extensive CNS and peripheral nervous system (PNS) involvement, widespread distribution of the exogenous gene may be required, a challenge to in vivo gene transfer strategies. Here, using lentiviral vectors (LVs), we efficiently transduced hematopoietic stem cells (HSCs) ex vivo and evaluated the potential of their progeny to target therapeutic genes to the CNS and PNS of transplanted mice and correct a neurodegenerative disorder, metachromatic leukodystrophy (MLD). We proved extensive repopulation of CNS microglia and PNS endoneurial macrophages by transgene-expressing cells. Intriguingly, recruitment of these HSC-derived cells was faster and more robust in MLD mice. By transplanting HSCs transduced with the arylsulfatase A gene, we fully reconstituted enzyme activity in the hematopoietic system of MLD mice and prevented the development of motor conduction impairment, learning and coordination deficits, and neuropathological abnormalities typical of the disease. Remarkably, ex vivo gene therapy had a significantly higher therapeutic impact than WT HSC transplantation, indicating a critical role for enzyme overexpression in the HSC progeny. These results indicate that transplantation of LV-transduced autologous HSCs represents a potentially efficacious therapeutic strategy for MLD and possibly other neurodegenerative disorders.     

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.     

Hematopoietic stem cell

Hematopoietic stem cells (HSCs) maintain themselves over cell divisions (self-renewal) and produce all kinds of blood cells (multi-potency). Depletion of these cells eventually causes hematopoietic failure, while deregulated HSC division causes development of myeloproliferative disorders and leukemias. HSCs can be prospectively purified to nearly homogeneity in mice, but such a high-level purification has not been achieved in humans. HSCs are localized to an anatomical place called 'niche'. Specialized osteoblasts arrayed on the endosteum of cavernous bone and sinusoidal endothelial cells located at the distant position from the endosteum are the two representative candidates of such an HSC niche. A number of adhesion molecules and signaling molecules are thought to comprise the niche-HSC synapse. HSCs divide only once in 1-2 months. Both environmental signaling from the niche and HSC-autonomous molecular programs contribute to the quiescent state of HSCs, which is essential for the maintenance of HSC self-renewal capacity and homeostasis of blood production.     

Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by cytoplasmic protein aggregates in the brain and spinal cord that include TAR-DNA binding protein 43 (TDP-43). TDP-43 is normally localized in the nucleus with roles in the regulation of gene expression, and pathological cytoplasmic aggregates are associated with depletion of nuclear protein. Here, we generated transgenic mice expressing human TDP-43 with a defective nuclear localization signal in the forebrain (hTDP-43-ΔNLS), and compared them with mice expressing WT hTDP-43 (hTDP-43-WT) to determine the effects of mislocalized cytoplasmic TDP-43 on neuronal viability. Expression of either hTDP-43-ΔNLS or hTDP-43-WT led to neuron loss in selectively vulnerable forebrain regions, corticospinal tract degeneration, and motor spasticity recapitulating key aspects of FTLD and primary lateral sclerosis. Only rare cytoplasmic phosphorylated and ubiquitinated TDP-43 inclusions were seen in hTDP-43-ΔNLS mice, suggesting that cytoplasmic inclusions were not required to induce neuronal death. Instead, neurodegeneration in hTDP-43 and hTDP-43-ΔNLS-expressing neurons was accompanied by a dramatic downregulation of the endogenous mouse TDP-43. Moreover, mice expressing hTDP-43-ΔNLS exhibited profound changes in gene expression in cortical neurons. Our data suggest that perturbation of endogenous nuclear TDP-43 results in loss of normal TDP-43 function(s) and gene regulatory pathways, culminating in degeneration of selectively vulnerable affected neurons.     

连接蛋白 Cx43参与冠状动脉平滑肌细胞的异质性促进冠脉再狭窄

目的：研究猪冠状动脉平滑肌细胞异质性与连接蛋白 Cx43关系,进一步阐明连接蛋白 Cx43在冠状动脉再狭窄发生中的作用。方法分别培养正常及支架术后猪原代冠状动脉平滑肌细胞,正常组应用血小板源生长因子（PDGF-BB）进行诱导,两组细胞分别进行电子显微镜观察细胞形态及结构变化,采用 Western Blotting 方法及实时荧光定量 PCR 测定 Cx43、Cx40、α-SMA、S100A4蛋白和 mRNA 表达;然后诱导组应用 Cx43阻断剂进行干预,再次检测上述指标变化。结果正常冠状动脉平滑肌细胞可见：纺锤型（spindle-shaped,S-SMC）和长菱形（rhomboid,R-SMC）两类,以 S-SMC 为主,其内 Cx40、α-SMA 有较高的蛋白和 mRNA 表达,而 Cx43表达较少;正常组经 PDGF-BB 诱导后细胞由 S-SMC 向 R-SMC 转变,同时伴随 Cx43表达上调,而 Cx40表达明显下降;通过反义 RNA 降低 Cx43表达,这种变化受抑制,并且 S-SMC 细胞保持原有形态,同时表达 a-肌动蛋白,支架组平滑肌细胞以 R-SMC 为主,Cx43、S100A4有较高的蛋白和 mRNA 表达。结论冠状动脉平滑肌细胞表型变化与连接蛋白 Cx43表达密切相关,连接蛋白 Cx43可能参与冠脉再狭窄的过程。