Identification of distinct conformations associated with monomers and fibril assemblies of mutant huntingtin.

The N-terminal fragments of mutant huntingtin (mHTT) misfold and assemble into oligomers, which ultimately bundle into insoluble fibrils. Conformations unique to various assemblies of mHTT remain unknown. Knowledge on the half-life of various multimeric structures of mHTT is also scarce. Using a panel of four new antibodies named PHP1-4, we have identified new conformations in monomers and assembled structures of mHTT. PHP1 and PHP2 bind to epitopes within the proline-rich domain (PRD), whereas PHP3 and PHP4 interact with motifs formed at the junction of polyglutamine (polyQ) and polyproline (polyP) repeats of HTT. The PHP1- and PHP2-reactive epitopes are exposed in fibrils of mHTT exon1 (mHTTx1) generated from recombinant proteins and mHTT assemblies, which progressively accumulate in the nuclei, cell bodies and neuropils in the brains of HD mouse models. Notably, electron microscopic examination of brain sections of HD mice revealed that PHP1- and PHP2-reactive mHTT assemblies are present in myelin sheath and in vesicle-like structures. Moreover, PHP1 and PHP2 antibodies block seeding and subsequent fibril assembly of mHTTx1 in vitro and in a cell culture model of HD. PHP3 and PHP4 bind to epitopes in full-length and N-terminal fragments of monomeric mHTT and binding diminishes as the mHTTx1 assembles into fibrils. Interestingly, PHP3 and PHP4 also prevent the aggregation of mHTTx1 in vitro highlighting a regulatory function for the polyQ-polyP motifs. These newly detected conformations may affect fibril assembly, stability and intercellular transport of mHTT.

The placental interleukin-6 signaling controls fetal brain development and behavior.

Epidemiological studies show that maternal immune activation (MIA) during pregnancy is a risk factor for autism. However, mechanisms for how MIA affects brain development and behaviors in offspring remain poorly described. To determine whether placental interleukin-6 (IL-6) signaling is required for mediating MIA on the offspring, we generated mice with restricted deletion of the receptor for IL-6 (IL-6Rα) in placental trophoblasts (Cyp19-Cre+;Il6rafl/fl), and tested offspring of Cyp19-Cre+;Il6rafl/fl mothers for immunological, pathological and behavioral abnormalities following induction of MIA. We reveal that MIA results in acute inflammatory responses in the fetal brain. Lack of IL-6 signaling in trophoblasts effectively blocks MIA-induced inflammatory responses in the placenta and the fetal brain. Furthermore, behavioral abnormalities and cerebellar neuropathologies observed in MIA control offspring are prevented in Cyp19-Cre+;Il6rafl/fl offspring. Our results demonstrate that IL-6 activation in placenta is required for relaying inflammatory signals to the fetal brain and impacting behaviors and neuropathologies relevant to neurodevelopmental disease.

Structural features and domain organization of huntingtin fibrils.

Misfolding and aggregation of huntingtin is one of the hallmarks of Huntington disease, but the overall structure of these aggregates and the mechanisms by which huntingtin misfolds remain poorly understood. Here we used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the structural features of huntingtin exon 1 (HDx1) containing 46 glutamine residues in its polyglutamine (polyQ) region. Despite some residual structuring in the N terminus, we find that soluble HDx1 is highly dynamic. Upon aggregation, the polyQ domain becomes strongly immobilized indicating significant tertiary or quaternary packing interactions. Analysis of spin-spin interactions does not show the close contact between same residues that is characteristic of the parallel, in-register structure commonly found in amyloids. Nevertheless, the same residues are still within 20 Å of each other, suggesting that polyQ domains from different molecules come into proximity in the fibrils. The N terminus has previously been found to take up a helical structure in fibrils. We find that this domain not only becomes structured, but that it also engages in tertiary or quaternary packing interactions. The existence of spin-spin interactions in this region suggests that such contacts could be made between N-terminal domains from different molecules. In contrast, the C-terminal domain is dynamic, contains polyproline II structure, and lacks pronounced packing interactions. This region must be facing away from the core of the fibrils. Collectively, these data provide new constraints for building structural models of HDx1 fibrils.

Modeling an autism risk factor in mice leads to permanent immune dysregulation.

Increasing evidence highlights a role for the immune system in the pathogenesis of autism spectrum disorder (ASD), as immune dysregulation is observed in the brain, periphery, and gastrointestinal tract of ASD individuals. Furthermore, maternal infection (maternal immune activation, MIA) is a risk factor for ASD. Modeling this risk factor in mice yields offspring with the cardinal behavioral and neuropathological symptoms of human ASD. In this study, we find that offspring of immune-activated mothers display altered immune profiles and function, characterized by a systemic deficit in CD4(+) TCRß(+) Foxp3(+) CD25(+) T regulatory cells, increased IL-6 and IL-17 production by CD4(+) T cells, and elevated levels of peripheral Gr-1(+) cells. In addition, hematopoietic stem cells from MIA offspring exhibit altered myeloid lineage potential and differentiation. Interestingly, repopulating irradiated control mice with bone marrow derived from MIA offspring does not confer MIA-related immunological deficits, implicating the peripheral environmental context in long-term programming of immune dysfunction. Furthermore, behaviorally abnormal MIA offspring that have been irradiated and transplanted with immunologically normal bone marrow from either MIA or control offspring no longer exhibit deficits in stereotyped/repetitive and anxiety-like behaviors, suggesting that immune abnormalities in MIA offspring can contribute to ASD-related behaviors. These studies support a link between cellular immune dysregulation and ASD-related behavioral deficits in a mouse model of an autism risk factor.

Exogenous leukemia inhibitory factor stimulates oligodendrocyte progenitor cell proliferation and enhances hippocampal remyelination.

New CNS neurons and glia are generated throughout adulthood from endogenous neural stem and progenitor cells. These progenitors can respond to injury, but their ability to proliferate, migrate, differentiate, and survive is usually insufficient to replace lost cells and restore normal function. Potentiating the progenitor response with exogenous factors is an attractive strategy for the treatment of nervous system injuries and neurodegenerative and demyelinating disorders. Previously, we reported that delivery of leukemia inhibitory factor (LIF) to the CNS stimulates the self-renewal of neural stem cells and the proliferation of parenchymal glial progenitors. Here we identify these parenchymal glia as oligodendrocyte (OL) progenitor cells (OPCs) and show that LIF delivery stimulates their proliferation through the activation of gp130 receptor signaling within these cells. Importantly, this effect of LIF on OPC proliferation can be harnessed to enhance the generation of OLs that express myelin proteins and reform nodes of Ranvier in the context of chronic demyelination in the adult mouse hippocampus. Our findings, considered together with the known beneficial effects of LIF on OL and neuron survival, suggest that LIF has both reparative and protective activities that make it a promising potential therapy for CNS demyelinating disorders and injuries.

Activation of the maternal immune system induces endocrine changes in the placenta via IL-6.

Activation of the maternal immune system in rodent models sets in motion a cascade of molecular pathways that ultimately result in autism- and schizophrenia-related behaviors in offspring. The finding that interleukin-6 (IL-6) is a crucial mediator of these effects led us to examine the mechanism by which this cytokine influences fetal development in vivo. Here we focus on the placenta as the site of direct interaction between mother and fetus and as a principal modulator of fetal development. We find that maternal immune activation (MIA) with a viral mimic, synthetic double-stranded RNA (poly(I:C)), increases IL-6 mRNA as well as maternally-derived IL-6 protein in the placenta. Placentas from MIA mothers exhibit increases in CD69+ decidual macrophages, granulocytes and uterine NK cells, indicating elevated early immune activation. Maternally-derived IL-6 mediates activation of the JAK/STAT3 pathway specifically in the spongiotrophoblast layer of the placenta, which results in expression of acute phase genes. Importantly, this parallels an IL-6-dependent disruption of the growth hormone-insulin-like growth factor (GH-IGF) axis that is characterized by decreased GH, IGFI and IGFBP3 levels. In addition, we observe an IL-6-dependent induction in pro-lactin-like protein-K (PLP-K) expression as well as MIA-related alterations in other placental endocrine factors. Together, these IL-6-mediated effects of MIA on the placenta represent an indirect mechanism by which MIA can alter fetal development.

Pulsed UV light inactivation of Salmonella enteritidis on eggshells and its effects on egg quality.

The majority of Salmonella Enteritidis outbreaks have been related to the consumption of raw or undercooked eggs or egg-containing foods. Therefore, the U.S. Department of Agriculture mandates egg washing for all graded eggs by use of a detergent solution and sanitizer. These agencies and the egg industry have been investigating alternative decontamination techniques, which could better serve the public, minimize costs, and benefit both the public and the industry. Pulsed UV light is an emerging technology that is used to inactivate microorganisms quickly. In this study, the effectiveness of pulsed UV light was evaluated for the decontamination of eggshells. Eggs inoculated with Salmonella Enteritidis on the top surface at the equator were treated with pulsed UV light 1 to 30 s, at a distance of 9.5 and 14.5 cm from the UV lamp in a laboratory-scale, pulsed UV light chamber. Three eggs were used per treatment in each repetition, except for quality measurements, which involved six eggs per treatment in each repetition. A maximum log reduction of 5.3 CFU/cm2 was obtained after a 20-s treatment at 9.5 cm below the UV lamp at a total dose of 23.6+/-0.1 J/cm2, without any visual damage to the egg. After a 30-s treatment at 9.5 and 14.5 cm, the temperature of eggshell surfaces increased by 16.3 and 13.3 degrees C, respectively. Energy usage increased up to 35.3+/-0.1 and 24.8+/-0.1 J/cm2, after 30-s treatments at 9.5 and 14.5 cm, respectively. The effect of pulsed UV light treatments on egg quality was also evaluated. Pulsed UV-light treatments for 3, 10, and 20s at either 9.5 or 14.5 cm did not change the albumen height, eggshell strength, or cuticle presence significantly (P<0.05). This study demonstrated that pulsed UV light has potential to decontaminate eggshell surfaces.

Maternal immune activation alters nonspatial information processing in the hippocampus of the adult offspring.

The observation that maternal infection increases the risk for schizophrenia in the offspring suggests that the maternal immune system plays a key role in the etiology of schizophrenia. In a mouse model, maternal immune activation (MIA) by injection of poly(I:C) yields adult offspring that display abnormalities in a variety of behaviors relevant to schizophrenia. As abnormalities in the hippocampus are a consistent observation in schizophrenia patients, we examined synaptic properties in hippocampal slices prepared from the offspring of poly(I:C)- and saline-treated mothers. Compared to controls, CA1 pyramidal neurons from adult offspring of MIA mothers display reduced frequency and increased amplitude of miniature excitatory postsynaptic currents. In addition, the specific component of the temporoammonic pathway that mediates object-related information displays increased sensitivity to dopamine. To assess hippocampal network function in vivo, we used expression of the immediate-early gene, c-Fos, as a surrogate measure of neuronal activity. Compared to controls, the offspring of poly(I:C)-treated mothers display a distinct c-Fos expression pattern in area CA1 following novel object, but not novel location, exposure. Thus, the offspring of MIA mothers may have an abnormality in modality-specific information processing. Indeed, the MIA offspring display enhanced discrimination in a novel object recognition, but not in an object location, task. Thus, analysis of object and spatial information processing at both synaptic and behavioral levels reveals a largely selective abnormality in object information processing in this mouse model. Our results suggest that altered processing of object-related information may be part of the pathogenesis of schizophrenia-like cognitive behaviors.

Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V(L)12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken beta-actin promoter. V(L)12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD.

Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35-40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1-MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.

Endothelin receptor B antagonists decrease glioma cell viability independently of their cognate receptor.

BACKGROUND: Endothelin receptor antagonists inhibit the progression of many cancers, but research into their influence on glioma has been limited. METHODS: We treated glioma cell lines, LN-229 and SW1088, and melanoma cell lines, A375 and WM35, with two endothelin receptor type B (ETRB)-specific antagonists, A-192621 and BQ788, and quantified viable cells by the capacity of their intracellular esterases to convert non-fluorescent calcein AM into green-fluorescent calcein. We assessed cell proliferation by labeling cells with carboxyfluorescein diacetate succinimidyl ester and quantifying the fluorescence by FACS analysis. We also examined the cell cycle status using BrdU/propidium iodide double staining and FACS analysis. We evaluated changes in gene expression by microarray analysis following treatment with A-192621 in glioma cells. We examined the role of ETRB by reducing its expression level using small interfering RNA (siRNA). RESULTS: We report that two ETRB-specific antagonists, A-192621 and BQ788, reduce the number of viable cells in two glioma cell lines in a dose- and time-dependent manner. We describe similar results for two melanoma cell lines. The more potent of the two antagonists, A-192621, decreases the mean number of cell divisions at least in part by inducing a G2/M arrest and apoptosis. Microarray analysis of the effects of A-192621 treatment reveals up-regulation of several DNA damage-inducible genes. These results were confirmed by real-time RT-PCR. Importantly, reducing expression of ETRB with siRNAs does not abrogate the effects of either A-192621 or BQ788 in glioma or melanoma cells. Furthermore, BQ123, an endothelin receptor type A (ETRA)-specific antagonist, has no effect on cell viability in any of these cell lines, indicating that the ETRB-independent effects on cell viability exhibited by A-192621 and BQ788 are not a result of ETRA inhibition. CONCLUSION: While ETRB antagonists reduce the viability of glioma cells in vitro, it appears unlikely that this effect is mediated by ETRB inhibition or cross-reaction with ETRA. Instead, we present evidence that A-192621 affects glioma and melanoma viability by activating stress/DNA damage response pathways, which leads to cell cycle arrest and apoptosis. This is the first evidence linking ETRB antagonist treatment to enhanced expression of DNA damage-inducible genes.

Intrabodies binding the proline-rich domains of mutant huntingtin increase its turnover and reduce neurotoxicity.

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

Maternal immune activation alters fetal brain development through interleukin-6.

Schizophrenia and autism are thought to result from the interaction between a susceptibility genotype and environmental risk factors. The offspring of women who experience infection while pregnant have an increased risk for these disorders. Maternal immune activation (MIA) in pregnant rodents produces offspring with abnormalities in behavior, histology, and gene expression that are reminiscent of schizophrenia and autism, making MIA a useful model of the disorders. However, the mechanism by which MIA causes long-term behavioral deficits in the offspring is unknown. Here we show that the cytokine interleukin-6 (IL-6) is critical for mediating the behavioral and transcriptional changes in the offspring. A single maternal injection of IL-6 on day 12.5 of mouse pregnancy causes prepulse inhibition (PPI) and latent inhibition (LI) deficits in the adult offspring. Moreover, coadministration of an anti-IL-6 antibody in the poly(I:C) model of MIA prevents the PPI, LI, and exploratory and social deficits caused by poly(I:C) and normalizes the associated changes in gene expression in the brains of adult offspring. Finally, MIA in IL-6 knock-out mice does not result in several of the behavioral changes seen in the offspring of wild-type mice after MIA. The identification of IL-6 as a key intermediary should aid in the molecular dissection of the pathways whereby MIA alters fetal brain development, which can shed new light on the pathophysiological mechanisms that predispose to schizophrenia and autism.

Leukemia inhibitory factor promotes neural stem cell self-renewal in the adult brain.

Although neural stem cells (NSCs) persist in various areas of the adult brain, their contribution to brain repair after injury is very limited. Treatment with exogenous growth factors can mitigate this limitation, suggesting that the brain environment is normally deficient in permissive cues and that it may be possible to stimulate the latent regenerative potential of endogenous progenitors with appropriate signals. We analyzed the effects of overexpressing the cytokine leukemia inhibitory factor (LIF) on adult neurogenesis in the normal brain. We found that LIF reduces neurogenesis in the olfactory bulb and subventricular zone by acting directly on NSCs. LIF appears to promote NSC self-renewal, preventing the emergence of more differentiated cell types. This ultimately leads to an expansion of the NSC pool. Our results have implications for the development of therapeutic strategies for brain repair and suggest that LIF may be useful, in combination with other factors, in promoting regeneration in the adult brain.

Behavioral stress and tumor progression.

BACKGROUND: A number of laboratories have reported a possible link between behavioral stress and cancer progression. Previously published findings demonstrated a stress-induced increase in tumor growth of implanted lymphosarcoma in C3H mice. Here, two mouse models were utilized to investigate whether stress alters the growth of solid tumors. MATERIALS AND METHODS: We developed a stress paradigm that involves alternating established stressors for 12 days. FVB mice implanted with melanoma were subjected to this stress protocol. We also attempted to duplicate Riley's finding. RESULTS: Our stress paradigm markedly increased serum corticosterone levels and thymus involution. No alteration in the growth of the melanoma tumors was observed. There was also no significant effect on lymphosarcoma progression using either our own or Riley's stress protocol. CONCLUSION: Under the conditions used in this study, strong behavioral stress did not influence tumor progression.

Leukemia inhibitory factor is a key regulator of astrocytic, microglial and neuronal responses in a low-dose pilocarpine injury model.

Insult to the central nervous system (CNS) induces many changes, including altered neurotransmitter expression, activation of astrocytes and microglia, neurogenesis and cell death. Cytokines and growth factors are candidates to be involved in astrocyte and microglial activation, and the up-regulation of glial fibrillary acidic protein (GFAP) is associated with brain damage. One of these candidates is leukemia inhibitory factor (LIF), a pro-inflammatory cytokine that is induced in astrocytes by brain damage or seizure. LIF also regulates expression of both neuropeptide Y (NPY) and galanin following peripheral nerve injury. To test the hypothesis that LIF regulates astrocyte, microglial and neuropeptide responses to a mild insult, we used a low-dose pilocarpine model to induce a brief seizure in LIF knock-out (KO) mice. Compared to wild type mice, the LIF KO mouse displays reduced astrocyte and microglial activation in the hippocampus. In addition, LIF KO mice display dramatically altered NPY, but not galanin, expression in response to injury. Thus, LIF is required for normal glial responses to brain damage, and, as in the periphery, LIF regulates NPY expression in the CNS.

Leukemia inhibitory factor promotes oligodendrocyte survival after spinal cord injury.

Injury to the mammalian spinal cord is accompanied by a delayed, secondary wave of oligodendrocyte apoptosis that arises several days after the initial injury. A strong candidate to support oligodendrocyte survival after spinal cord injury is the pleiotropic cytokine, leukemia inhibitory factor (LIF). In vitro, LIF potentiates the differentiation and survival of oligodendrocyte precursors. LIF can also prevent oligodendrocyte apoptosis in response to either growth factor removal or cytotoxic challenge. More recently, in vivo studies have demonstrated that LIF is effective in preventing oligodendrocyte death in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). We therefore asked whether systemic delivery of LIF could ameliorate oligodendrocyte death in a mouse model of spinal cord injury. We have found that daily administration of LIF (25 microg/kg/day) promotes oligodendrocyte survival after spinal cord injury. Interestingly however, this effect does not appear to be mediated by a direct action of LIF on the oligodendrocyte but rather via an ancillary cell type, which results in augmented expression of another trophic factor capable of supporting oligodendrocyte survival, insulin-like growth factor 1 (IGF-1).

Endothelin receptor B inhibition triggers apoptosis and enhances angiogenesis in melanomas.

Endothelin receptor B (ETRB or EDNRB) is overexpressed in most human melanomas and is proposed to provide a marker of melanoma progression. We have shown previously that inhibition of ETRB leads to increased human melanoma cell death in vitro and in vivo, resulting in shrinkage of tumors grown in immunocompromised mice. In the present work, we analyzed the effects of ETRB inhibition on 10 human melanoma cell lines derived from tumors at distinct stages of progression. Our observations suggest that the ETRB antagonist BQ788 induces apoptosis most effectively in metastatic melanoma cells. Microarray analysis shows that BQ788 treatment leads to a reduction in the expression of the survival factor BCL-2A1 and the DNA repair factor poly(ADP-ribose) polymerase 3 that is more pronounced in cells derived from metastatic than primary melanoma. Decreased cell viability was observed to correlate with reduction in ETRB expression, and reduction in ETRB protein levels by small interfering RNA led to an increase in cell death. Interestingly, reduction of ETRB expression by BQ788 was accompanied by a strong induction of VEGF expression and repression of the angiogenic suppressor gravin. These changes in gene expression correlated with increased angiogenesis in tumors injected with ETRB antagonist in vivo. Taken together, our observations suggest that ETRB may provide a potential therapeutic target in high-grade melanomas and identify candidate pathways that may be implicated in the regulation of cell survival and tumor progression associated with ETRB signaling.

Targeted overexpression of leukemia inhibitory factor to preserve myocardium in a rat model of postinfarction heart failure.

OBJECTIVE: Myocardial infarction leads to cardiomyocyte loss. The cytokine leukemia inhibitory factor regulates the differentiation and growth of embryonic and adult heart tissue. This study examined the effects of gene transfer of leukemia inhibitory factor in infarcted rat hearts. METHODS: Lewis rats underwent ligation of the left anterior descending coronary artery and direct injection of adenovirus encoding leukemia inhibitory factor (n = 10) or null transgene as control (n = 10) into the myocardium bordering the ischemic area. A sham operation group (n = 10) underwent thoracotomy without ligation. After 6 weeks, the following parameters were evaluated: cardiac function with a pressure-volume conductance catheter, left ventricular geometry and architecture by histologic methods; myocardial fibrosis by Masson trichrome staining, apoptosis by terminal deoxynucleotidal transferase-mediated deoxyuridine triphosphate nick-end labeling assay, and cardiomyocyte size by immunofluorescence. RESULTS: Rats with overexpression of leukemia inhibitory factor had more preserved myocardium and less fibrosis in both the infarct and its border zone. The border zone in leukemia inhibitory factor-treated animals contained fewer apoptotic nuclei (1.6% +/- 0.1% vs 3.3% +/- 0.2%, P < .05) than that in control animals and demonstrated cardiomyocytes with larger cross-sectional areas (910 +/- 60 microm 2 vs 480 +/- 30 microm 2 , P < .05). Leukemia inhibitory factor-treated animals had increased left ventricular wall thickness (2.1 +/- 0.1 mm vs 1.8 +/- 0.1 mm, P < .05) and less dilation of the left ventricular cavity (237 +/- 22 microL vs 301 +/- 16 microL, P < .05). They also had improved cardiac function, as measured by maximum change in pressure over time (3950 +/- 360 mm Hg/s vs 2750 +/- 230 mm Hg/s, P < .05) and the slopes of the maximum change in pressure over time-end-diastolic volume relationship (68 +/- 5 mm Hg/[s . microL] vs 46 +/- 6 mm Hg/[s . microL], P < .05) and the preload recruitable stroke work relationship (89 +/- 10 mm Hg vs 44 +/- 4 mm Hg, P < .05). CONCLUSIONS: Myocardial gene transfer of leukemia inhibitory factor preserved cardiac tissue, geometry, and function after myocardial infarction in rats.

Activation of the IkappaB kinase complex and nuclear factor-kappaB contributes to mutant huntingtin neurotoxicity.

Transcriptional dysregulation by mutant huntingtin (Htt) protein has been implicated in the pathogenesis of Huntington's disease (HD). We find that cultured cells expressing mutant Htt and striatal cells from HD transgenic mice have elevated nuclear factor-kappaB (NF-kappaB) activity. Furthermore, NF-kappaB is concentrated in the nucleus of neurons in the brains of HD transgenic mice. In inducible PC12 cells and in HD transgenic mice, mutant Htt activates the IkappaB kinase complex (IKK), a key regulator of NF-kappaB. Activation of IKK is likely mediated by direct interaction with mutant Htt, because the expanded polyglutamine stretch and adjacent proline-rich motifs in mutant Htt interact with IKKgamma, a regulatory subunit of IKK. Activation of IKK may also influence the toxicity of mutant Htt, because expression of IKKgamma promotes aggregation and nuclear localization of mutant Htt exon-1. Moreover, in acute striatal slice cultures, inhibition of IKK activity with an N-terminally truncated form of IKKgamma blocks mutant Htt-induced toxicity in medium-sized spiny neurons (MSNs). In addition, blocking degradation of NF-kappaB inhibitors with a dominant-negative ubiquitin ligase beta-transducin repeat-containing protein also reduces the toxicity of mutant Htt in MSNs. Therefore, aberrant NF-kappaB activation may contribute to the neurodegeneration induced by mutant Htt.