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Hata S Koyama S Kawahara H Doi N Maeda T Toyama-Sorimachi N Abe K Suzuki K Sorimachi H 《The Journal of biological chemistry》2006,281(16):11214-11224
Calpain is a Ca2+-regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast two-hybrid screening identified several nCL-2-interacting molecules. Of these, the beta-subunit of coatomer complex (beta-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, beta-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca2+-ionophore stimulation caused the proteolysis of beta-COP near the linker region, resulting in the dissociation of beta-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein. 相似文献
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Yoko Kudo-Sakamoto Hiroshi Akazawa Kaoru Ito Jiro Takano Masamichi Yano Chizuru Yabumoto Atsuhiko T. Naito Toru Oka Jong-Kook Lee Yasushi Sakata Jun-ichi Suzuki Takaomi C. Saido Issei Komuro 《The Journal of biological chemistry》2014,289(28):19408-19419
Enzymatic proteolysis by calpains, Ca2+-dependent intracellular cysteine proteases, has been implicated in pathological processes such as cellular degeneration or death. Here, we investigated the role of calpain activation in the hearts subjected to myocardial infarction. We produced myocardial infarction in Cast−/− mice deficient for calpastatin, the specific endogenous inhibitory protein for calpains, and Cast+/+ mice. The activity of cardiac calpains in Cast+/+ mice was not elevated within 1 day but showed a gradual elevation after 7 days following myocardial infarction, which was further pronounced in Cast−/− mice. Although the prevalence of cardiomyocyte death was indistinguishable between Cast−/− and Cast+/+ mice, Cast−/− mice exhibited profound contractile dysfunction and chamber dilatation and showed a significant reduction in survival rate after myocardial infarction as compared with Cast+/+ mice. Notably, immunofluorescence revealed that at 28 days after myocardial infarction, calpains were activated in cardiomyocytes exclusively at the border zone and that Cast−/− mice showed higher intensity and a broader extent of calpain activation at the border zone than Cast+/+ mice. In the border zone of Cast−/− mice, pronounced activation of calpains was associated with a decrease in N-cadherin expression and up-regulation of molecular markers for cardiac hypertrophy and fibrosis. In cultured rat neonatal cardiomyocytes, calpain activation by treatment with ionomycin induced cleavage of N-cadherin and decreased expression levels of β-catenin and connexin 43, which was attenuated by calpain inhibitor. These results thus demonstrate that activation of calpains disassembles cell-cell adhesion at intercalated discs by degrading N-cadherin and thereby promotes left ventricular remodeling after myocardial infarction. 相似文献
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Calpains constitute a family of intracellular Ca(2+)-regulated cysteine proteases that are indispensable in the regulation of a wide variety of cellular functions. The improper activation of calpain causes lethality or various disorders, such as muscular dystrophies and tumor formation. nCL-2/calpain 8 is predominantly expressed in the stomach, where it appears to be involved in membrane trafficking in the gastric surface mucus cells (pit cells). Although the primary structure of nCL-2 is quite similar to that of the ubiquitous m-calpain large subunit, the enzymatic properties of nCL-2 have never been reported. Here, to characterize nCL-2, the recombinant protein was prepared using an Escherichia coli expression system and purified to homogeneity. nCL-2 was stably produced as a soluble and active enzyme without the conventional calpain regulatory subunit (30K). Purified nCL-2 showed Ca(2+)-dependent activity, with half-maximal activity at about 0.3 mM Ca(2+), similar to that of m-calpain, whereas its optimal pH and temperature were comparatively low. Immunoprecipitation analysis revealed that nCL-2 exists in both monomeric and homo-oligomeric forms, but not as a heterodimer with 30K or 30K-2, and that the oligomerization occurs through domains other than the 5EF-hand domain IV, most probably through domain III, suggesting a novel regulatory system for nCL-2. 相似文献
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Ojima K Ono Y Ottenheijm C Hata S Suzuki H Granzier H Sorimachi H 《Journal of molecular biology》2011,407(3):439-449
Mutations in CAPN3/Capn3, which codes for skeletal muscle-specific calpain-3/p94 protease, are responsible for limb-girdle muscular dystrophy type 2A. Using “knock-in” (referred to as Capn3CS/CS) mice, in which the endogenous calpain-3 is replaced with a mutant calpain-3:C129S, which is a proteolytically inactive but structurally intact calpain-3, we demonstrated in our previous studies that loss of calpain-3 protease activity causes muscular dystrophy [Ojima, K. et al. (2010) J. Clin. Invest. 120, 2672-2683]. However, compared to Capn3-null (Capn3−/−) mice, Capn3CS/CS mice showed less severe dystrophic symptoms. This suggests that calpain-3 also has a non-proteolytic function. This study aimed to elucidate the non-proteolytic functions of calpain-3 through comparison of Capn3CS/CS mice with Capn3−/− mice. We found that calpain-3 is a component of the sarcoplasmic reticulum (SR), and that calpain-3 interacts with, but does not proteolyze, typical SR components such as ryanodine receptor and calsequestrin. Furthermore, Capn3CS/CS mice showed that the nonenzymatic role of calpain-3 is required for proper Ca2+ efflux from the SR to cytosol during muscle contraction. These results indicate that calpain-3 functions as a nonenzymatic element for the Ca2+ efflux machinery in the SR, rather than as a protease. Thus, defects in the nonenzymatic function of calpain-3 must also be involved in the pathogenesis of limb-girdle muscular dystrophy type 2A. 相似文献
8.
F M Menzies M Garcia-Arencibia S Imarisio N C O'Sullivan T Ricketts B A Kent M V Rao W Lam Z W Green-Thompson R A Nixon L M Saksida T J Bussey C J O'Kane D C Rubinsztein 《Cell death and differentiation》2015,22(3):433-444
Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington''s disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington''s disease.Huntington''s disease (HD) is a currently incurable, autosomal dominant neurodegenerative disease resulting from the expansion of the trinucleotide (CAG) repeat region of the huntingtin (HTT) IT15 gene, encoding huntingtin protein (Htt). In mutant Htt, the polyglutamine tract encoded by this region contains over 35 glutamines and the length of the tract correlates inversely with the age of disease onset, with longer tracts resulting in earlier onset (reviewed in Imarisio et al.1). HD is one of the 10 trinucleotide repeat disorders resulting from expansions of polyglutamine tracts in different proteins. These expansions cause disease by conferring toxic gain-of-function properties onto the mutant proteins. Hence, one strategy that has been considered for HD and related diseases is to find ways of decreasing the levels of the mutant protein, for instance by harnessing the cell''s capacity to degrade such aggregate-prone proteins via (macro)autophagy.2, 3, 4, 5 Autophagy involves the engulfment of cytoplasmic contents by double-membraned autophagosomes, which then traffic to lysosomes where their contents are degraded. Mutant huntingtin, some other polyglutamine expanded proteins like mutant ataxin 3, and proteins like tau (which mediates toxicity in Alzheimer''s disease and related dementias) are autophagy substrates and their clearance can be enhanced in Drosophila and mouse models by autophagy upregulation, which also reduces their toxicity.2, 3, 4,6Calpains are a family of calcium-activated cysteine proteases (reviewed in Ono and Sorimachi7) that inhibit autophagy. Strategies that reduce calpain activity in cell culture increase autophagy and decrease levels of autophagy substrates, like mutant Htt. These effects are likely to be mediated by Gsα, a heterotrimeric G-protein subunit which is activated by calpain cleavage. Similar to calpain inhibition, siRNA knockdown of Gsα, or chemical inhibition by NF449, induces autophagy and decreases the number of aggregates resulting from the overexpression of exon-1 Htt with an expanded polyglutamine repeat region (HttQ74) in cell culture models.8 In addition to this mechanism of autophagy upregulation by calpains, the core autophagy protein ATG5 has also been demonstrated to be cleaved and inactivated by calpains,9,10 suggesting that calpains may act on a number of substrates to negatively regulate autophagy.In mammals, the two most abundantly expressed calpains are μ-calpain and m-calpain, which differ in their affinity for calcium and therefore the calcium concentration required for their activation. As well as being regulated by calcium, they are also controlled by an endogenous inhibitor, calpastatin (CAST). Drosophila have four forms of calpain:11 CalpA and CalpB are the conventional calpains formed by a recent duplication in the Drosophila insect lineage, CalpC is also an evolutionarily recent, but not highly conserved duplication (data not shown) and is thought to be catalytically inactive,11 and CalpD (SOL) is a member of the unconventional family of calpains. Drosophila does not appear to have any obvious orthologs of CAST.A role for calpains in HD has been investigated previously. Following observations that shorter Htt fragments are more toxic than full-length Htt,12 it was demonstrated that Htt can be cleaved by both caspases13 and calpains14 to generate these toxic, short fragments. Blocking Htt cleavage by calpains by mutating their calpain cleavage sites decreases Htt aggregation and toxicity.15 In addition, calpain activation has been shown to be increased in HD patients compared with controls.14In this study, we have investigated a role for calpain activity as a modulator of autophagy in both Drosophila and mouse models of HD. To avoid confounding effects from alterations in cleavage of Htt by calpain, we have used models expressing short fragments of Htt, which do not contain calpain cleavage sites and correspond to the shortest fragments of huntingtin seen in patients.16 We demonstrate that knockdown of CalpA in Drosophila is sufficient to both reduce the number of Htt aggregates and the toxicity associated with the expression of the mutant protein. Importantly, we show that these effects are autophagy-dependent. Furthermore, we show that overexpression of CAST in mice results in enhanced autophagy and improves locomotor function and delays tremor onset in a mouse model of HD, as well as decreasing the number of Htt aggregates seen in the brain. We extended the analysis of CAST overexpressing mice to investigate the possible adverse effects from long-term calpain inhibition or autophagy upregulation but did not observe any obvious deleterious effects. 相似文献
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Erwin Reiling Martijn E. T. Dollé Sameh A. Youssef Moonsook Lee Bhawani Nagarajah Marianne Roodbergen Piet de With Alain de Bruin Jan H. Hoeijmakers Jan Vijg Harry van Steeg Paul Hasty 《PloS one》2014,9(4)
Ku80 and DNA-PKCS are both involved in the repair of double strand DNA breaks via the nonhomologous end joining (NHEJ) pathway. While ku80−/− mice exhibit a severely reduced lifespan and size, this phenotype is less pronounced in dna-pkcs−/− mice. However, these observations are based on independent studies with varying genetic backgrounds. Here, we generated ku80−/−, dna-pkcs−/− and double knock out mice in a C57Bl6/J*FVB F1 hybrid background and compared their lifespan, end of life pathology and mutation frequency in liver and spleen using a lacZ reporter. Our data confirm that inactivation of Ku80 and DNA-PKCS causes reduced lifespan and bodyweights, which is most severe in ku80−/− mice. All mutant mice exhibited a strong increase in lymphoma incidence as well as other aging-related pathology (skin epidermal and adnexal atrophy, trabacular bone reduction, kidney tubular anisokaryosis, and cortical and medullar atrophy) and severe lymphoid depletion. LacZ mutation frequency analysis did not show strong differences in mutation frequencies between knock out and wild type mice. The ku80−/− mice had the most severe phenotype and the Ku80-mutation was dominant over the DNA-PKCS-mutation. Presumably, the more severe degenerative effect of Ku80 inactivation on lifespan compared to DNA-PKCS inactivation is caused by additional functions of Ku80 or activity of free Ku70 since both Ku80 and DNA-PKCS are essential for NHEJ. 相似文献
11.
Sabine Krueger Anja Bernhardt Thomas Kalinski Martin Baldensperger Michael Zeh Anne Teller Daniela Adolf Thomas Reinheckel Albert Roessner Doerthe Kuester 《PloS one》2013,8(7)
Helicobacter pylori are responsible for the induction of chronic gastric inflammation progressing to atrophy, metaplasia, and gastric cancer. The overexpression of Cathepsin X/Z (Ctsz) in H. pylori-infected mucosa and gastric cancer is mediated predominantly by an augmented migration of ctsz−/−positive macrophages and the up-regulation of Ctsz in tumor epithelium. To explore the Ctsz-function in the context of chronic inflammation and the development of preneoplastic lesions, we used Ctsz-deficient mice in a H. pylori gastritis model. Ctsz
−/− and wild-type (wt) mice were infected with H. pylori strain SS1. The mice were sacrificed at 24, 36, and 50 weeks post infection (wpi). The stomach was removed, and gastric strips were snap-frozen or embedded and stained with H&E. Tissue sections were scored for epithelial lesions and inflammation. Ki-67 and F4/80 immunostaining were used to measure epithelial cell proliferation and macrophage infiltration, respectively. The upregulation of compensating cathepsins and cytokines were confirmed by Western blotting and quantitative RT-PCR. SS1-infected wt and ctsz
−/− mice showed strong inflammation, foveolar hyperplasia, atrophy, and cystically-dilated glands. However, at 50 wpi, ctsz
−/− mice developed significantly more severe spasmolytic polypeptide-expressing metaplasia (SPEM), showed enhanced epithelial proliferation, and higher levels of infiltrating macrophages. Induction of cytokines was higher and significantly prolonged in ctsz
−/− mice compared to wt. Ctsz deficiency supports H. pylori-dependent development of chronic gastritis up to metaplasia, indicating a protective, but not proteolytic, function of Ctsz in inflammatory gastric disease. 相似文献
12.
Alicia De Maria Yanrong Shi Nalin M. Kumar Steven Bassnett 《The Journal of biological chemistry》2009,284(20):13542-13550
In animal models, the dysregulated activity of calcium-activated proteases,
calpains, contributes directly to cataract formation. However, the
physiological role of calpains in the healthy lens is not well defined. In
this study, we examined the expression pattern of calpains in the mouse lens.
Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7
were expressed in lens fiber cells. Using controlled lysis, depth-dependent
expression profiles for each calpain were obtained. These indicated that,
unlike calpain 1, 2, and 7, which were most abundant in cells near the lens
surface, calpain 3 expression was strongest in the deep cortical region of the
lens. We detected calpain activities in vitro and showed that
calpains were active in vivo by microinjecting fluorogenic calpain
substrates into cortical fiber cells. To identify endogenous calpain
substrates, membrane/cytoskeleton preparations were treated with recombinant
calpain, and cleaved products were identified by two-dimensional difference
electrophoresis/mass spectrometry. Among the calpain substrates identified by
this approach was αII-spectrin. An antibody that specifically recognized
calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved
in vivo, late in fiber cell differentiation, at or about the time
that lens organelles are degraded. The generation of the calpain-specific
spectrin cleavage product was not observed in lens tissue from calpain 3-null
mice, indicating that calpain 3 is uniquely activated during lens fiber
differentiation. Our data suggest a role for calpains in the remodeling of the
membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium
dependence of the founder members of the family, the ubiquitously expressed
enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain
family includes more than a dozen members with sequence relatedness to the
catalytic subunits of calpain 1 and 2. Calpains have a modular domain
architecture. By convention, the family is subdivided into classical and
nonclassical calpains, according to the presence or absence, respectively, of
a calcium-binding penta-EF-hand module in domain IV of the protein
(1). Classical calpains include
calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7,
10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an
essential role for calpains during embryonic development. Knock-out of the
small regulatory subunit (Capn4) results in embryonic lethality
(2,
3). Similarly, inactivation of
the Capn2 gene blocks development between the morula and blastocyst
stage (4). In humans, mutations
in CAPN3 underlie limb-girdle muscular dystrophy-2A, and
polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus
(5,
6).Even under conditions of calcium overload, where calpains are presumably
activated maximally, only a subset (<5%) of cellular proteins are
hydrolyzed (7). Calpains
typically cleave their substrates at a limited number of sites to generate
large polypeptide fragments that, in many cases, retain bioactivity. Thus,
under physiological conditions, calpains probably participate in the
regulation of protein function rather than in non-specific protein
degradation.More than 100 proteins have been shown to serve as calpain substrates
in vitro, including cytoskeletal proteins
(8), signal transduction
molecules (9), ion channels
(10), and receptors
(11). In vivo,
calpains are believed to function in myoblast fusion
(12), long term potentiation
(13), and cellular mobility
(14). Unregulated calpain
activity, secondary to intracellular calcium overload, is associated with
several pathological conditions, including Alzheimer disease
(15), animal models of
cataract (16), myocardial
(17), and cerebral ischemia
(18).In addition to their domain structure, calpains are often classified
according to their tissue expression patterns. Calpain 1, 2, and 10 are widely
expressed in mammalian tissues, but other members of the calpain family show
tissue-specific expression patterns. Calpain 8, for example, is a
stomach-specific calpain (19),
whereas expression of calpain 9 is restricted to tissues of the digestive
tract (20). The expression of
calpain 3 was originally thought to be limited to skeletal muscle
(21), but splice variants of
calpain 3 have since been detected in a range of tissues. At least 12 isoforms
of calpain 3 have been described in rodents
(22), of which several are
expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88
(retina) (23).Calpains have been studied intensively in the ocular lens because of their
suspected involvement in lens opacification (cataract). Calpain-mediated
proteolysis of lens crystallin proteins causes increased light scatter
(24). Unregulated activation
of calpains is observed in rodent cataract models
(25), where calpain-mediated
degradation of crystallin proteins
(26) and cytoskeletal elements
(27) is commonly observed.
Calpain inhibitors are effective in delaying or preventing cataract in
vitro (28,
29) and in vivo
(30,
31).It is likely, however, that calpains have important physiological roles in
the lens beyond their involvement in tissue pathology. Terminal
differentiation of lens fiber cells involves a series of profound
morphological and biochemical transformations. For example, differentiating
lens fiber cells undergo an enormous (>100-fold) increase in cell length,
accompanied by extensive remodeling of the plasma membrane system
(32). Early in the
differentiation process, fusion pores are established between cells, as
neighboring fibers are incorporated into the lens syncytium
(33). A later stage of fiber
cell differentiation involves the dissolution of all intracellular organelles,
a process that is thought to eliminate light-scattering particles from the
light path and contribute to the transparency of the tissue
(34). Any or all of these
phenomena might require the developmentally regulated activation of calpains.
This is consistent with our previous observation that in calpain 3 knock-out
mice the transition zone is altered, suggesting a change in the
differentiation program
(35).In the current study, therefore, we examined the depth-dependent expression
pattern and activity of calpains in the mouse lens. Fluorogenic substrates
were microinjected into the intact lens to visualize calpain activity
directly, and proteomic approaches were used to identify endogenous calpain
substrates. The cleavage pattern of one of these, αII-spectrin, was
examined in detail. Immunocytochemical and immunoblot analysis with wild type
and calpain 3-null lenses indicated that αII-spectrin is a specific
calpain 3 substrate in maturing lens fiber cells. Together, the data suggest
that calpains are activated relatively late in fiber cell differentiation and
may contribute to the remodeling of the membrane cytoskeleton that accompanies
fiber cell maturation. 相似文献
13.
Rami A. Namas John Bartels Rosemary Hoffman Derek Barclay Timothy R. Billiar Ruben Zamora Yoram Vodovotz 《PloS one》2013,8(7)
We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO2
−/NO3
− data from “middle-aged” (6–8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for “young” (2–3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging. 相似文献
14.
Li Jiang Yuxiao Wei Cecinio C. Ronquillo Robert E. Marc Bradley K. Yoder Jeanne M. Frederick Wolfgang Baehr 《The Journal of biological chemistry》2015,290(20):12765-12778
Anterograde intraflagellar transport (IFT) employing kinesin-2 molecular motors has been implicated in trafficking of photoreceptor outer segment proteins. We generated embryonic retina-specific (prefix “emb”) and adult tamoxifen-induced (prefix “tam”) deletions of KIF3a and IFT88 in adult mice to study photoreceptor ciliogenesis and protein trafficking. In embKif3a−/− and in embIft88−/− mice, basal bodies failed to extend transition zones (connecting cilia) with outer segments, and visual pigments mistrafficked. In contrast, tamKif3a−/− and tamIft88−/− photoreceptor axonemes disintegrated slowly post-induction, starting distally, but rhodopsin and cone pigments trafficked normally for more than 2 weeks, a time interval during which the outer segment is completely renewed. The results demonstrate that visual pigments transport to the retinal outer segment despite removal of KIF3 and IFT88, and KIF3-mediated anterograde IFT is responsible for photoreceptor transition zone and axoneme formation. 相似文献
15.
Alejandra Rangel Noelia Madro?al Agnès Gruart i. Massó Rosalina Gavín Franc Llorens Lauro Sumoy Juan María Torres José María Delgado-García José Antonio Del Río 《PloS one》2009,4(10)
Background
Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrPsc) of the natural cellular prion protein (PrPc) encoded by the Prnp gene. Although several roles have been attributed to PrPc, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrPc studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation.Methodology/Principal Findings
Here we explore the role of PrPc expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrPc-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABAA and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice.Conclusions/Significance
Present results demonstrate that PrPc is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABAA and AMPA-Kainate neurotransmission. New PrPc functions have recently been described, which point to PrPc as a target for putative therapies in Alzheimer''s disease. However, our results indicate that a “gain of function” strategy in Alzheimer''s disease, or a “loss of function” in prionopathies, may impair PrPc function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies. 相似文献16.
David A. Potter Jennifer S. Tirnauer Richard Janssen Dorothy E. Croall Christina N. Hughes Kerry A. Fiacco James W. Mier Masatoshi Maki Ira M. Herman 《The Journal of cell biology》1998,141(3):647-662
Previous studies suggest that the Ca2+-dependent proteases, calpains, participate in remodeling of the actin cytoskeleton during wound healing and are active during cell migration. To directly test the role that calpains play in cell spreading, several NIH-3T3– derived clonal cell lines were isolated that overexpress the biological inhibitor of calpains, calpastatin. These cells stably overexpress calpastatin two- to eightfold relative to controls and differ from both parental and control cell lines in morphology, spreading, cytoskeletal structure, and biochemical characteristics. Morphologic characteristics of the mutant cells include failure to extend lamellipodia, as well as abnormal filopodia, extensions, and retractions. Whereas wild-type cells extend lamellae within 30 min after plating, all of the calpastatin-overexpressing cell lines fail to spread and assemble actin-rich processes. The cells genetically altered to overexpress calpastatin display decreased calpain activity as measured in situ or in vitro. The ERM protein ezrin, but not radixin or moesin, is markedly increased due to calpain inhibition. To confirm that inhibition of calpain activity is related to the defect in spreading, pharmacological inhibitors of calpain were also analyzed. The cell permeant inhibitors calpeptin and MDL 28, 170 cause immediate inhibition of spreading. Failure of the intimately related processes of filopodia formation and lamellar extension indicate that calpain is intimately involved in actin remodeling and cell spreading. 相似文献
17.
Laura Magnaghi-Jaulin Anne Marcilhac Mireille Rossel Christian Jaulin Yves Benyamin Fabrice Raynaud 《Chromosoma》2010,119(3):267-274
Calpains form a family of Ca2+-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform
remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that
Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2
depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the
loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may
play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion. 相似文献
18.
19.
Yuning Xiong Sahil Khanna Adrienne L. Grzenda Olga F. Sarmento Phyllis A. Svingen Gwen A. Lomberk Raul A. Urrutia William A. Faubion Jr. 《The Journal of biological chemistry》2012,287(41):34372-34385
Inducible gene expression underlies the epigenetically inherited differentiation program of most immune cells. We report that the promoter of the FOXP3 gene possesses two distinct functional states: an “off state” mediated by the polycomb histone methyltransferase complex and a histone acetyltransferase-dependent “on state.” Regulating these states is the presence of a Kruppel-like factor (KLF)-containing Polycomb response element. In the KLF10−/− mouse, the FOXP3 promoter is epigenetically silenced by EZH2 (Enhancer of Zeste 2)-mediated trimethylation of Histone 3 K27; thus, impaired FOXP3 induction and inappropriate adaptive T regulatory cell differentiation results in vitro and in vivo. The epigenetic transmittance of adaptive T regulatory cell deficiency is demonstrated throughout more than 40 generations of mice. These results provide insight into chromatin remodeling events key to phenotypic features of distinct T cell populations. 相似文献
20.
Venkateswaran Subramanian Jessica J. Moorleghen Anju Balakrishnan Deborah A. Howatt Athar H. Chishti Haruhito A. Uchida 《PloS one》2013,8(8)