IL-6 Mediated Degeneration of Forebrain GABAergic Interneurons and Cognitive Impairment in Aged Mice through Activation of Neuronal NADPH Oxidase

Background

Multiple studies have shown that plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) are elevated in patients with important and prevalent adverse health conditions, including atherosclerosis, diabetes, obesity, obstructive sleep apnea, hypertension, and frailty. Higher plasma levels of IL-6, in turn, increase the risk of many conditions associated with aging including age-related cognitive decline. However, the mechanisms underlying this association between IL-6 and cognitive vulnerability remain unclear.

Methods and Findings

We investigated the role of IL-6 in brain aging in young (4 mo) and aged (24 mo) wild-type C57BL6 and genetically-matched IL-6^−/− mice, and determined that IL-6 was necessary and sufficient for increased neuronal expression of the superoxide-producing immune enzyme, NADPH-oxidase, and this was mediated by non-canonical NFκB signaling. Furthermore, superoxide production by NADPH-oxidase was directly responsible for age-related loss of parvalbumin (PV)-expressing GABAergic interneurons, neurons essential for normal information processing, encoding, and retrieval in hippocampus and cortex. Targeted deletion of IL-6 or elimination of superoxide by chronic treatment with a superoxide-dismutase mimetic prevented age-related loss of PV-interneurons and reversed age-related cognitive deficits on three standard tests of spatial learning and recall.

Conclusions

Present results indicate that IL-6 mediates age-related loss of critical PV-expressing GABAergic interneurons through increased neuronal NADPH-oxidase-derived superoxide production, and that rescue of these interneurons preserves cognitive performance in aging mice, suggesting that elevated peripheral IL-6 levels may be directly and mechanistically linked to long-lasting cognitive deficits in even normal older individuals. Further, because PV-interneurons are also selectively affected by commonly used anesthetic agents and drugs, our findings imply that IL-6 levels may predict adverse CNS effects in older patients exposed to these compounds through specific derangements in inhibitory interneurons, and that therapies directed at lowering IL-6 may have cognitive benefits clinically.     

Dysfunction of Orbitofrontal GABAergic Interneurons Leads to Impaired Reversal Learning in a Mouse Model of Obsessive-Compulsive Disorder

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Functional Maturation of hPSC-Derived Forebrain Interneurons Requires an Extended Timeline and Mimics Human Neural Development

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Inhibitory Postsynaptic Currents Induced by Activation of Interneurons in Cultured Rat Hippocampal Neurons

Using the patch-clamp technique in the whole-cell configuration, we studied the characteristics of a series of action potentials (APs) induced by a 500-msec-long current pulse applied to a pre-synaptic unit, as well as the kinetic characteristics of post-synaptic currents (PSCs) evoked by the APs in a post-synaptic unit, in synaptically connected pairs of cultured hippocampal neurons. Presynaptic inhibitory units were identified as GABA-ergic interneurons; they were divided into two groups according to the size of the soma and the number of processes. The kinetic characteristics of PSCs, which were induced in the post-synaptic neuron by a series of the APs generated in the pre-synaptic cell, demonstrated a certain dependence on the morphological characteristics of these cells. In interneurons with large-sized somata, the kinetics of the currents were more fast, and the reversal potential was close to the equilibrium Cl potential. In interneurons with small-sized somata, currents were slower, and the reversal potential was shifted. We conclude that under conditions of culturing, a pre-synaptic cell not only directly provokes the development of PSC in a post-synaptic neuron and determines the amplitude of this current but also significantly influences the kinetics of this current. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 116–123, March–April, 2005.     

Fgfr1 Inactivation in the Mouse Telencephalon Results in Impaired Maturation of Interneurons Expressing Parvalbumin

Fibroblast growth factors (Fgfs) and their receptors (Fgfr) are expressed in the developing and adult CNS. Previous studies demonstrated a decrease in cortical interneurons and locomotor hyperactivity in mice with a conditional Fgfr1 deletion generated in radial glial cells during midneurogenesis (Fgfr1^f/f;hGfapCre+). Here, we report earlier and more extensive inactivation of Fgfr1 in neuroepithelial cells of the CNS (Fgfr1^f/f;NesCre+). Similar to findings in Fgfr1^f/f;hGfapCre+ mice, parvalbumin positive (PV+) cortical interneurons are also decreased in the neocortex of Fgfr1f/f;NesCre+ mice when compared to control littermates (Fgfr1f/f). Fgfr1f/f;NesCre+ embryos do not differ from controls in the initial specification of GABAergic cells in the ganglionic eminence (GE) as assessed by in situ hybridization for Dlx2, Mash1 and Nkx2. Equal numbers of GABAergic neuron precursors genetically labeled with green fluorescent protein (GFP) were observed at P0 in Fgfr1^f/f;hGfapCre+;Gad1-GFP mutant mice. However, fewer GFP+ and GFP+/PV+ interneurons were observed in these mutants at adulthood, indicating that a decrease in cortical interneuron markers is occurring postnatally. Fgfr1 is expressed in cortical astrocytes in the postnatal brain. To test whether the astrocytes of mice lacking Fgfr1 are less capable of supporting interneurons, we co-cultured wild type Gad1-GFP+ interneuron precursors isolated from the medial GE (MGE) with astrocytes from Fgfr1f/f control or Fgfr1^f/f;hGfapCre+ mice. Interneurons grown on Fgfr1 deficient astrocytes had small soma size and fewer neurites per cell, but no differences in cell survival. Decreased soma size of Gad67 immunopositive interneurons was also observed in the cortex of adult Fgfr1^f/f;NesCre+ mice. Our data indicate that astrocytes from Fgfr1 mutants are impaired in supporting the maturation of cortical GABAergic neurons in the postnatal period. This model may elucidate potential mechanisms of impaired PV interneuron maturation relevant to neuropsychiatric disorders that develop in childhood and adolescence.     

AKT1E17K Is Oncogenic in Mouse Lung and Cooperates with Chemical Carcinogens in Inducing Lung Cancer

The hotspot AKT1^E17K mutation in the pleckstrin homology domain of AKT1 occurs in approximately 0.6–2% of human lung cancers. Recently, we have demonstrated that AKT1^E17K transforms immortalized human bronchial cells. Here by use of a transgenic Cre-inducible murine strain in the wild type Rosa26 (R26) locus (R26-AKT1^E17K mice) we demonstrate that AKT1^E17K is a bona-fide oncogene and plays a role in the development of lung cancer in vivo. In fact, we report that mutant AKT1^E17K induces bronchial and/or bronchiolar hyperplastic lesions in murine lung epithelium, which progress to frank carcinoma at very low frequency, and accelerates tumor formation induced by chemical carcinogens. In conclusion, AKT1^E17K induces hyperplasia of mouse lung epithelium in vivo and cooperates with urethane to induce the fully malignant phenotype.     

Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changes

Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells beta-actin rather than alpha-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization.     

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S- and M-phase cyclins (A and B), securin, and geminin (13, 25, 31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G₁/S- and M-phase progression and inducing rereplication (6, 21, 25, 31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1^m/m) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutagenesis screen designed to identify genes that are necessary for normal morphological development in embryos (1, 8, 9). Subsequent studies showed that the insertion was located between the first and second exons of the emi1 gene (2). The morphological defects in emi1^m/m embryos at 2 days postfertilization (p.f.) are described in the public access zebrafish model organism database (http://zfin.org). Briefly, abnormalities in emi1^m/m embryos can be identified as early as 20 h p.f. and include slightly smaller heads and a lack of ventral curving of the posterior presomitic mesoderm. By 25 h p.f., the tail is more ventrally curved than in normal embryos, and increased cell death is observed throughout the central nervous system. Mutant embryos have circulating blood cells, although the onset of circulation is delayed. We became interested in this mutant because it harbors defects in the numbers and morphology of granulocytes, an important myeloid cell type within the innate immune system.There is evidence that EMI1 may function in cancer pathogenesis, and a variety of human tumors express this factor very highly, although in some cases this may be a consequence of elevated proliferation rates (11, 18). In fact, the human homologue of emi1 resides within chromosome 6q25, a region often deleted in leukemia, which, together with the cell cycle-regulatory role of EMI1, suggested that this factor may also function as a tumor suppressor whose loss of function could promote genetic instability. Thus, in addition to investigating the role of zebrafish emi1 in zebrafish development, with particular emphasis on hematopoiesis, we examined mammalian cells to identify mechanisms that may be important in EMI1-related malignant transformation and explored a putative tumor suppressor role for this cell cycle-regulatory protein.     

PRC1 Cooperates with CLASP1 to Organize Central Spindle Plasticity in Mitosis

During cell division, chromosome segregation is governed by the interaction of spindle microtubules with the kinetochore. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, microtubule-bundling protein PRC1, and Aurora B kinase complex. However, the precise role of PRC1 in central spindle organization has remained elusive. Here we show that PRC1 recruits CLASP1 to the central spindle at early anaphase onset. CLASP1 belongs to a conserved microtubule-binding protein family that mediates the stabilization of overlapping microtubules of the central spindle. PRC1 physically interacts with CLASP1 and specifies its localization to the central spindle. Repression of CLASP1 leads to sister-chromatid bridges and depolymerization of spindle midzone microtubules. Disruption of PRC1-CLASP1 interaction by a membrane-permeable peptide abrogates accurate chromosome segregation, resulting in sister chromatid bridges. These findings reveal a key role for the PRC1-CLASP1 interaction in achieving a stable anti-parallel microtubule organization essential for faithful chromosome segregation. We propose that PRC1 forms a link between stabilization of CLASP1 association with central spindle microtubules and anti-parallel microtubule elongation.To ensure that each daughter cell receives the full complement of the genome in each cell division, chromosomes move poleward, and non-kinetochore fibers become bundled at the onset of anaphase, initiating assembly of the central spindle, a set of anti-parallel microtubules that serves to concentrate key regulators of cytokinesis (1–3). Chromosomal passengers are a group of evolutionarily conserved proteins that orchestrates chromosome segregation and central spindle plasticity (4, 5). This protein complex containing Aurora B, Survivin, INCENP, and Borealin is relocated from the kinetochore to the central spindle upon anaphase onset (5–9). Perturbation of their function results in defects in metaphase chromosome alignment, chromosome segregation, and cytokinesis (10).Among the central spindle maintenance components, only two have been reported to mediate the microtubule bundling in the central spindle. One is centralspindlin, a heterotetramer containing CeMKLP1/ZEN-4 and RhoGAP/CYK-4 (11), and the other one is an evolutionarily conserved protein, PRC1 (also named Feo in fruit fry, Ase1 in yeast, and MAP65 in plant cells). PRC1 is a non-motor microtubule-binding and -bundling protein in human cells originally identified as a Cdc2 substrate essential for cytokinesis (12, 13). Similar microtubule regulatory activities have been reported in yeast, fruit fly, and plant cells. It is well known that overexpression of wild type PRC1 in HeLa cells can result in thick microtubule bundles in cells at interphase (13). Bundling activity of PRC1, as well as centralspindlin, is required for the organization of the central spindle as well as for the successful progression of cytokinesis. PRC1 molecules accumulate on the midline of a central spindle with the cell cycle progression to anaphase. As a non-motor microtubule-binding protein, transportation of PRC1 to the midline is promoted by its association to kinesin, KIF4A, and timing of this progression is controlled by the dephosphorylation of Thr-481 on PRC1 when the cell exits metaphase by phosphatase Cdc14 (14). Our recent study shows that prevention of the phosphorylation of PRC1 at Thr-470 causes an inhibition in PRC1 oligomerization in vitro and an aberrant organization of central spindle in vivo, suggesting that this phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle (15).Spatiotemporal regulation of microtubule organization and dynamics is responsible for the mitotic apparatus such as the central spindle. However, it has remained elusive as to how the central spindle microtubule organization and dynamics are regulated. There are large varieties of microtubule-associated proteins responsible for regulation of the dynamic behavior of microtubules and microtubule-mediated transport. Among these, proteins that associate with the tips of microtubules are called +TIPs, for “plus-end tracking proteins.” These proteins have been shown to be important in different organisms and cellular systems (16). Using yeast two-hybrid assay, CLASPs were identified as interacting partners of the CLIPs and characterized as new +TIP proteins (17).The microtubule-binding protein CLASP is emerging as an important microtubule regulator in the formation of the mitotic apparatus (18–22). CLASP is required for promoting plus-end growth of spindle microtubules in prometaphase (23). Although the molecular mechanisms underlying its regulation of microtubule dynamics remain elusive, it is generally believed that CLASP orchestrates microtubule dynamics via its physical interacting with EB1, CLIP170, and microtubules (17, 24).To delineate the molecular function of PRC1 in central spindle organization and spatiotemporal regulation, we carried out a new search for PRC1-interacting proteins. Our studies show that PRC1 physically interacts with CLASP1, and the two proteins cooperate in the organization of the central spindle. Our studies provide a novel regulatory mechanism in which the PRC1 complex operates central spindle organization in mitosis.     

mGluR1-Mediated Excitation of Cerebellar GABAergic Interneurons Requires Both G Protein-Dependent and Src–ERK1/2-Dependent Signaling Pathways

Stimulation of type I metabotropic glutamate receptors (mGluR1/5) in several neuronal types induces slow excitatory responses through activation of transient receptor potential canonical (TRPC) channels. GABAergic cerebellar molecular layer interneurons (MLIs) modulate firing patterns of Purkinje cells (PCs), which play a key role in cerebellar information processing. MLIs express mGluR1, and activation of mGluR1 induces an inward current, but its precise intracellular signaling pathways are unknown. We found that mGluR1 activation facilitated spontaneous firing of mouse cerebellar MLIs through an inward current mediated by TRPC1 channels. This mGluR1-mediated inward current depends on both G protein-dependent and -independent pathways. The nonselective protein tyrosine kinase inhibitors genistein and AG490 as well as the selective extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors PD98059 and SL327 suppressed the mGluR1-mediated current responses. Following G protein blockade, the residual mGluR1-mediated inward current was significantly reduced by the selective Src tyrosine kinase inhibitor PP2. In contrast to cerebellar PCs, GABA_B receptor activation in MLIs did not alter the mGluR1-mediated inward current, suggesting that there is no cross-talk between mGluR1 and GABA_B receptors in MLIs. Thus, activation of mGluR1 facilitates firing of MLIs through the TRPC1-mediated inward current, which depends on not only G protein-dependent but also Src–ERK1/2-dependent signaling pathways, and consequently depresses the excitability of cerebellar PCs.     

Allele-Specific Non-CpG Methylation of the Nf1 Gene during Early Mouse Development

Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Our examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation was not found in later stages of embryo development or in adult tissue. Our results suggest that maternal-specific de novo non-CpG methylation has occurred sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele has undergone site-specific active demethylation. Our data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides.     

Morphological Diversity of GABAergic and Cholinergic Interneurons in the Striatal Dorsolateral and Ventromedial Regions of Rats

The striatum plays a fundamental role in sensorimotor and cognitive functions of the body, and different sub-regions control different physiological functions. The striatal interneurons play important roles in the striatal function, yet their specific functions are not clearly elucidated so far. The present study aimed to investigate the morphological properties of the GABAergic interneurons expressing neuropeptide Y (NPY), calretinin (Cr), and parvalbumin (Parv) as well as the cholinergic interneurons expressing choline acetyltransferase (ChAT) in the striatal dorsolateral (DL) and ventromedial (VM) regions of rats using immunohistochemistry and Western blot. The present results showed that the somatic size of Cr+ was the smallest, while ChAT+ was the largest among the four types of interneurons. There was no regional difference in neuronal somatic size of all types of interneurons. Cr+ and Parv+ neurons were differentially distributed in the striatum. Moreover, Parv+ had the longest primary dendrites in the DL region, while NPY+ had the longest ones in the VM region of striatum. But there was regional difference in the length of primary dendrites of Parv. The numbers of primary dendrites of Parv+ were the largest in both DL and VM regions of striatum. Both Cr+ and Parv+ primary dendrites displayed regional difference in the striatum. Western blot further confirmed the regional differences in the protein expression level of Cr and Parv. Hence, the present study indicates that GABAergic and cholinergic interneurons might be involved in different physiological functions based on their morphological and distributional diversity in different regions of the rat striatum.     

小鼠短暂前脑缺血海马中半胱天冬酶-３酶原表达的变化

通过测定脑缺血再灌注时海马中半胱天冬酶-3酶原(procaspase-3)的表达变化, 从细胞凋亡的角度探讨脑缺血再灌注损伤的分子生物学机制及procaspase-3的活化机制.将C57BL/6N小鼠随机分为假手术组(正常对照组)、缺血再灌注组(I/R组), 后者夹闭双侧颈总动脉20 min后再通血流, 建立前脑缺血再灌注模型, 分别于再灌注6 h、12 h、24 h和48 h取海马.采用蛋白免疫印迹(Western blotting)方法检测海马中procaspase-3的表达变化.结果显示, 12 h I/R及24hI/R组海马中总procaspase-3水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),24 h I/R组海马中去磷酸化水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),而各组procaspase-3磷酸化水平与假手术组相比差异无统计学意义.结果提示, 脑缺血再灌注损伤诱发procaspase-3表达增加,其中procaspase-3去磷酸化水平高明显, 提示脑缺血再灌注损伤可能诱发procaspase-3去磷酸化, 继而促进procaspase-3转化为活性形式.     

Inhibition of Fumonisin B1 Cytotoxicity by Nanosilicate Platelets during Mouse Embryo Development

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B₁ (FB₁), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB₁. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB₁ only, a very low residual level of FB₁ in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB₁ and NSP, suggesting a high alleviation effect of NSP on FB₁ in vivo. Furthermore, FB₁ treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB₁ to animals.