Pentraxin 3 promotes airway inflammation in experimental asthma.

BACKGROUND: Pentraxin 3 (PTX3) regulates multiple aspects of innate immunity and tissue inflammation. Recently, it has been reported that PTX3 deficiency enhances interleukin (IL)-17A-dominant pulmonary inflammation in an ovalbumin (OVA)-induced mouse asthma model. However, whether PTX3 treatment would provide protection against allergic airway inflammation has not been clearly elucidated. The goal of this study was to further investigate the effect of recombinant PTX3 administration on the phenotype of asthma. METHODS: C57BL/6 J mice were sensitized and challenged with OVA to induce eosinophilic asthma model, as well as sensitized with OVA plus LPS and challenged with OVA to induce neutrophilic asthma model. We evaluated effect of recombinant PTX3 on asthma phenotype through both asthma models. The bronchoalveolar lavage fluid (BALF) inflammatory cells and cytokines, airway hyperresponsiveness, and pathological alterations of the lung tissues were assessed. RESULTS: In both eosinophilic and neutrophilic asthma models, PTX3 treatment provoked airway hyperresponsiveness, concomitant with increased inflammatory cytokines IL-4, IL-17, eotaxin, and transforming growth factor (TGF)-ß1 and aggravated airway accumulation of inflammatory cells, especially eosinophils and neutrophils. In histological analysis of the lung tissue, administration of PTX3 promoted inflammatory cells infiltration, mucus production, and collagen deposition. In addition, PTX3 also significantly enhanced STAT3 phosphorylation in lung tissue. CONCLUSION: Our results show that exogenous PTX3 can exacerbate multiple asthmatic features by promoting both eosinophils and neutrophils lung infiltration and provide new evidence to better understand the complex role of PTX3 in allergic airway inflammation.

Autoimmune encephalitis mediated by B-cell response against N-methyl-d-aspartate receptor.

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a neuropsychiatric disease characterized by an antibody-mediated autoimmune response against NMDAR. Recent studies have shown that anti-NMDAR antibodies are involved in the pathophysiology of the disease. However, the upstream immune and inflammatory processes responsible for this pathogenic response are still poorly understood. Here, we immunized mice against the region of NMDA receptor containing the N368/G369 amino acids, previously implicated in a pathogenic response. This paradigm induced encephalopathy characterized by blood-brain barrier opening, periventricular T2-MRI hyperintensities and IgG deposits into the brain parenchyma. Two weeks after immunization, mice developed clinical symptoms reminiscent of encephalitis: anxiety- and depressive-like behaviours, spatial memory impairment (without motor disorders) and increased sensitivity to seizures. This response occurred independently of overt T-cell recruitment. However, it was associated with B220+ (B cell) infiltration towards the ventricles, where they differentiated into CD138+ cells (plasmocytes). Interestingly, these B cells originated from peripheral lymphoid organs (spleen and cervical lymphoid nodes). Finally, blocking the B-cell response using a depleting cocktail of antibodies reduced the severity of symptoms in encephalitis mice. This study demonstrates that the B-cell response can lead to an autoimmune reaction against NMDAR that drives encephalitis-like behavioural impairments. It also provides a relevant platform for dissecting encephalitogenic mechanisms in an animal model, and enables the testing of therapeutic strategies targeting the immune system in anti-NMDAR encephalitis.

Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract.

Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

Nesfatin-1, a potent anorexic agent, decreases exploration and induces anxiety-like behavior in rats without altering learning or memory.

The anorectic neuropeptide nesfatin-1 has recently been characterized as a potential mood regulator, but the accurate effect of nesfatin-1 on anxiety and learning and memory behavior and the possible mechanisms remains unknown. In the present study, to test the hypothesis that nesfatin-1 might affect the anxiety-like and learning and memory behaviors in rats via ERK/CREB/BDNF pathway, nesfatin-1 was administered intraperitoneally to rats with the doses (10, 20, 40µg/kg), and the behavioral performance was tested using the open field task, the Morris water maze (MWM), and the Y maze. Moreover, the protein expression of brain-derived neurotrophic factor (BDNF), total and phosphorylated-ERK in the hippocampus and the prefrontal cortex (PFC) were evaluated. The results showed that chronic administration of nesfatin-1 could decrease the moving distance, the duration in the center, and the frequencies of rearing and grooming in the open field task, decrease the moving distance, frequency, and the preference index of new arm in the Y maze, although there was no significant difference of the performance in the MWM task among groups. Furthermore, 3 weeks' consecutive administration of nesfatin-1 resulted in the decrease of protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC. These results provided evidence that exogenous nesfatin-1 could decrease exploration and induce anxiety-like behavior in rats, the mechanism of which might be related to the reduced protein expression of BDNF and phosphorylated-ERK in the hippocampus and the PFC.

α-Synuclein modifies mutant huntingtin aggregation and neurotoxicity in Drosophila.

Protein misfolding and aggregation is a major hallmark of neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Until recently, the consensus was that each aggregation-prone protein was characteristic of each disorder [α-synuclein (α-syn)/PD, mutant huntingtin (Htt)/HD, Tau and amyloid beta peptide/AD]. However, growing evidence indicates that aggregation-prone proteins can actually co-aggregate and modify each other's behavior and toxicity, suggesting that this process may also contribute to the overlap in clinical symptoms across different diseases. Here, we show that α-syn and mutant Htt co-aggregate in vivo when co-expressed in Drosophila and produce a synergistic age-dependent increase in neurotoxicity associated to a decline in motor function and life span. Altogether, our results suggest that the co-existence of α-syn and Htt in the same neuronal cells worsens aggregation-related neuropathologies and accelerates disease progression.

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease.

Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.

Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons.

BACKGROUND: Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) subunits, suggesting these ion channels contribute to Artn-induced sensitivity. Here we have used gene expression, immunolabeling, patch clamp electrophysiology and behavioral testing assays to investigate the link between Artn, nicotinic subunit expression and thermal hypersensitivity. RESULTS: Reverse transcriptase-PCR validation showed increased levels of mRNAs encoding the nAChR subunits α3 (13.3-fold), ß3 (4-fold) and ß4 (7.7-fold) in trigeminal ganglia and α3 (4-fold) and ß4 (2.8-fold) in dorsal root ganglia (DRG) of ART-OE mice. Sensory ganglia of ART-OE mice had increased immunoreactivity for nAChRα3 and exhibited increased overlap in labeling with GFRα3-positive neurons. Patch clamp analysis of back-labeled cutaneous afferents showed that while the majority of nicotine-evoked currents in DRG neurons had biophysical and pharmacological properties of α7-subunit containing nAChRs, the Artn-induced increase in α3 and ß4 subunits resulted in functional channels. Behavioral analysis of ART-OE and wildtype mice showed that Artn-induced thermal hyperalgesia can be blocked by mecamylamine or hexamethonium. Complete Freund's adjuvant (CFA) inflammation of paw skin, which causes an increase in Artn in the skin, also increased the level of nAChR mRNAs in DRG. Finally, the increase in nAChRs transcription was not dependent on the Artn-induced increase in TRPV1 or TRPA1 in ART-OE mice since nAChRs were elevated in ganglia of TRPV1/TRPA1 double knockout mice. CONCLUSIONS: These findings suggest that Artn regulates the expression and composition of nAChRs in GFRα3 nociceptors and that these changes contribute to the thermal hypersensitivity that develops in response to Artn injection and perhaps to inflammation.

Aß dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies.

Dimers of Aß (amyloid ß-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aß](DiY) (dityrosine cross-linked Aß). For comparison, we used the Aß monomer and a design dimer cross-linked by replacement of Ser²6 with cystine [AßS26C]2. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aß](DiY) and [AßS26C]2 have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aß monomers. Our results indicate that the link between Aß dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

The Hsp70/90 cochaperone, Sti1, suppresses proteotoxicity by regulating spatial quality control of amyloid-like proteins.

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of ß-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

Different ataxin-3 amyloid aggregates induce intracellular Ca(2+) deregulation by different mechanisms in cerebellar granule cells.

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Tat-collapsin response mediator protein 2 (CRMP2) increases the survival of neurons after NMDA excitotoxity by reducing the cleavage of CRMP2.

Collapsin response mediator protein 2 (CRMP2) is a brain-specific multifunctional adaptor protein involved in neuronal polarity and axonal guidance. Our previous results showed CRMP2 may be involved in the hypoxic preconditioning and ischemic injury, but the mechanism was not clear. This study explored whether CRMP2 was involved in NMDA-induced neural death, and the possible mechanism. Western blot analysis demonstrated that NMDA reduced the phosphorylation of CRMP2 and inspired the cleavage of CRMP2. Also, it was detected that NMDA treatment did not affect the phosphorylation of CRMP2 in early stage (<6 h). Over-expression of CRMP2 aggravated the NMDA-induced injury, suggesting the vital role of CRMP2 in excitotoxicity. Tat-CRMP2 was designed to provide the cleavage site of calpain. Thiazolyl blue tetrazolium bromide assay, Hoechst33342/Propidium Iodide staining and Western blot assay showed that Tat-CRMP2 pretreatment increased cell viability compared with the control group against NMDA exposure by decreasing the cleavage of CRMP2. In conclusion, these studies indicated that cleavage of CRMP2 plays an important role involved in the NMDA-induced injury. The cleavage of CRMP2 may be a promising target for excitatory amino acid-related ischemic and hypoxic injury.

New hope for therapy in neurodegenerative diseases.

The use of genetic screens to define cellular pathways that regulate neurodegenerative disease proteins has emerged as a powerful strategy to identify potential therapeutic targets for these disorders. Using cross-species genetic screens, Park et al. recently identified RAS-MAPK-MSK1 as a cellular pathway that modulates levels of the polyglutamine-containing protein ATXN1 and its subsequent toxicity in SCA1.

Isolation of cytotoxic compounds from the seeds of Crataegus pinnatifida.

AIM: To study the chemical constituents and bioactivity of the seeds of Crataegus pinnatifida. METHODS: The chemical constituents were isolated and purified by macroporous adsorptive resin D101, silica gel, and ODS column chromatography, and preparative HPLC. Their structures were elucidated on the basis of spectroscopic methods. In addition, the cytotoxic activities of compounds 1-4 were investigated on OPM2 and RPMI-8226 cells. RESULTS: Four compounds were obtained and their structures were identified as (7S, 8S)-4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-(hydroxymethyl)ethoxy]-3, 5-dimethoxybenzaldehyde (1), (+)-balanophonin (2), erythro-guaiacylglycerol-ß-coniferyl aldehyde ether (3), buddlenol A (4). CONCLUSION: Compound 1 is a novel norlignan, while compounds 1-4 exhibited marginal inhibition on the proliferation of OPM2 and RPMI-8226 cells.

RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1.

Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.

Identification of NUB1 as a suppressor of mutant Huntington toxicity via enhanced protein clearance.

Huntington's disease is caused by expanded CAG repeats in HTT, conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model, and 6 exhibited activity consistent with the in vitro screening results. Among these, negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment, interferon-ß lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus, we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease.

CART peptide induces neuroregeneration in stroke rats.

Utilizing a classic stroke model in rodents, middle cerebral artery occlusion (MCAo), we describe a novel neuroregenerative approach using the repeated intranasal administration of cocaine- and amphetamine-regulated transcript (CART) peptide starting from day 3 poststroke for enhancing the functional recovery of injured brain. Adult rats were separated into two groups with similar infarction sizes, measured by magnetic resonance imaging on day 2 after MCAo, and were treated with CART or vehicle. The CART treatment increased CART level in the brain, improved behavioral recovery, and reduced neurological scores. In the subventricular zone (SVZ), CART enhanced immunolabeling of bromodeoxyuridine, a neural progenitor cell marker Musashi-1, and the proliferating cell nuclear antigen, as well as upregulated brain-derived neurotrophic factor (BDNF) mRNA. AAV-GFP was locally applied to the SVZ to examine migration of SVZ cells. The CART enhanced migration of GFP(+) cells from SVZ toward the ischemic cortex. In SVZ culture, CART increased the size of neurospheres. The CART-mediated cell migration from SVZ explants was reduced by anti-BDNF blocking antibody. Using (1)H-MRS (proton magnetic resonance spectroscopy), increases in N-acetylaspartate levels were found in the lesioned cortex after CART treatment in stroke brain. Cocaine- and amphetamine-regulated transcript increased the expression of GAP43 and fluoro-ruby fluorescence in the lesioned cortex. In conclusion, our data suggest that intranasal CART treatment facilitates neuroregeneration in stroke brain.

A genome-scale RNA-interference screen identifies RRAS signaling as a pathologic feature of Huntington's disease.

A genome-scale RNAi screen was performed in a mammalian cell-based assay to identify modifiers of mutant huntingtin toxicity. Ontology analysis of suppressor data identified processes previously implicated in Huntington's disease, including proteolysis, glutamate excitotoxicity, and mitochondrial dysfunction. In addition to established mechanisms, the screen identified multiple components of the RRAS signaling pathway as loss-of-function suppressors of mutant huntingtin toxicity in human and mouse cell models. Loss-of-function in orthologous RRAS pathway members also suppressed motor dysfunction in a Drosophila model of Huntington's disease. Abnormal activation of RRAS and a down-stream effector, RAF1, was observed in cellular models and a mouse model of Huntington's disease. We also observe co-localization of RRAS and mutant huntingtin in cells and in mouse striatum, suggesting that activation of R-Ras may occur through protein interaction. These data indicate that mutant huntingtin exerts a pathogenic effect on this pathway that can be corrected at multiple intervention points including RRAS, FNTA/B, PIN1, and PLK1. Consistent with these results, chemical inhibition of farnesyltransferase can also suppress mutant huntingtin toxicity. These data suggest that pharmacological inhibition of RRAS signaling may confer therapeutic benefit in Huntington's disease.

p10, the N-terminal domain of p35, protects against CDK5/p25-induced neurotoxicity.

Cyclin-dependent kinase 5(CDK5) in complex with its activator, p35 (protein of 35 kDa), is essential for early neurodevelopment in mammals. However, endogenous cleavage of p35 to p25 is associated with neuron death and neurodegenerative disease. Here we show that a peptide (p10') encoding the N-terminal domain of p35 protects against CDK5/p25-induced toxicity in neurons. p10' also prevented the death of neurons treated with the neurotoxin, 1-methyl-4-phenylpyridinium (MPP(+)), which induces conversion of endogenous p35 to p25, and Parkinson disease (PD)-like symptoms in animals. MPP(+) induces CDK5/p25-dependent phosphorylation of peroxiredoxin 2 (Prx2), resulting in inhibition of its peroxireductase activity and accumulation of reactive oxygen species (ROS). We found that p10' expression inhibited both Prx2 phosphorylation and ROS accumulation in neurons. In addition, p10' inhibited the p25-induced appearance of antigen of the Ki67 antibody (Ki67) and phosphohistone H2AX (γH2AX), classic markers of cell cycle activity and DNA double-strand breakage, respectively, associated with neuron death. Our results suggest that p10 (protein of 10 kDa) is a unique prosurvival domain in p35, essential for normal CDK5/p35 function in neurons. Loss of the p10 domain results in CDK5/p25 toxicity and neurodegeneration in vivo.