A lentiviral toolkit to monitor airway epithelial cell differentiation using bioluminescence.

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC, and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures.NEW & NOTEWORTHY Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence.

Immune Surveillance in Clinical Regression of Preinvasive Squamous Cell Lung Cancer.

Before squamous cell lung cancer develops, precancerous lesions can be found in the airways. From longitudinal monitoring, we know that only half of such lesions become cancer, whereas a third spontaneously regress. Although recent studies have described the presence of an active immune response in high-grade lesions, the mechanisms underpinning clinical regression of precancerous lesions remain unknown. Here, we show that host immune surveillance is strongly implicated in lesion regression. Using bronchoscopic biopsies from human subjects, we find that regressive carcinoma in situ lesions harbor more infiltrating immune cells than those that progress to cancer. Moreover, molecular profiling of these lesions identifies potential immune escape mechanisms specifically in those that progress to cancer: antigen presentation is impaired by genomic and epigenetic changes, CCL27-CCR10 signaling is upregulated, and the immunomodulator TNFSF9 is downregulated. Changes appear intrinsic to the carcinoma in situ lesions, as the adjacent stroma of progressive and regressive lesions are transcriptomically similar. SIGNIFICANCE: Immune evasion is a hallmark of cancer. For the first time, this study identifies mechanisms by which precancerous lesions evade immune detection during the earliest stages of carcinogenesis and forms a basis for new therapeutic strategies that treat or prevent early-stage lung cancer.See related commentary by Krysan et al., p. 1442.This article is highlighted in the In This Issue feature, p. 1426.

Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization.

Streptococcus pneumoniae (Spn) is a common cause of respiratory infection, but also frequently colonizes the nasopharynx in the absence of disease. We used mass cytometry to study immune cells from nasal biopsy samples collected following experimental human pneumococcal challenge in order to identify immunological mechanisms of control of Spn colonization. Using 37 markers, we characterized 293 nasal immune cell clusters, of which 7 were associated with Spn colonization. B cell and CD8+CD161+ T cell clusters were significantly lower in colonized than in non-colonized subjects. By following a second cohort before and after pneumococcal challenge we observed that B cells were depleted from the nasal mucosa upon Spn colonization. This associated with an expansion of Spn polysaccharide-specific and total plasmablasts in blood. Moreover, increased responses of blood mucosal associated invariant T (MAIT) cells against in vitro stimulation with pneumococcus prior to challenge associated with protection against establishment of Spn colonization and with increased mucosal MAIT cell populations. These results implicate MAIT cells in the protection against pneumococcal colonization and demonstrate that colonization affects mucosal and circulating B cell populations.

mTORC1 amplifies the ATF4-dependent de novo serine-glycine pathway to supply glycine during TGF-ß1-induced collagen biosynthesis.

The differentiation of fibroblasts into a transient population of highly activated, extracellular matrix (ECM)-producing myofibroblasts at sites of tissue injury is critical for normal tissue repair. Excessive myofibroblast accumulation and persistence, often as a result of a failure to undergo apoptosis when tissue repair is complete, lead to pathological fibrosis and are also features of the stromal response in cancer. Myofibroblast differentiation is accompanied by changes in cellular metabolism, including increased glycolysis, to meet the biosynthetic demands of enhanced ECM production. Here, we showed that transforming growth factor-ß1 (TGF-ß1), the key pro-fibrotic cytokine implicated in multiple fibrotic conditions, increased the production of activating transcription factor 4 (ATF4), the transcriptional master regulator of amino acid metabolism, to supply glucose-derived glycine to meet the amino acid requirements associated with enhanced collagen production in response to myofibroblast differentiation. We further delineated the signaling pathways involved and showed that TGF-ß1-induced ATF4 production depended on cooperation between canonical TGF-ß1 signaling through Smad3 and activation of mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). ATF4, in turn, promoted the transcription of genes encoding enzymes of the de novo serine-glycine biosynthetic pathway and glucose transporter 1 (GLUT1). Our findings suggest that targeting the TGF-ß1-mTORC1-ATF4 axis may represent a novel therapeutic strategy for interfering with myofibroblast function in fibrosis and potentially in other conditions, including cancer.

Deciphering the genomic, epigenomic, and transcriptomic landscapes of pre-invasive lung cancer lesions.

The molecular alterations that occur in cells before cancer is manifest are largely uncharted. Lung carcinoma in situ (CIS) lesions are the pre-invasive precursor to squamous cell carcinoma. Although microscopically identical, their future is in equipoise, with half progressing to invasive cancer and half regressing or remaining static. The cellular basis of this clinical observation is unknown. Here, we profile the genomic, transcriptomic, and epigenomic landscape of CIS in a unique patient cohort with longitudinally monitored pre-invasive disease. Predictive modeling identifies which lesions will progress with remarkable accuracy. We identify progression-specific methylation changes on a background of widespread heterogeneity, alongside a strong chromosomal instability signature. We observed mutations and copy number changes characteristic of cancer and chart their emergence, offering a window into early carcinogenesis. We anticipate that this new understanding of cancer precursor biology will improve early detection, reduce overtreatment, and foster preventative therapies targeting early clonal events in lung cancer.

Analysis of RNA Expression Profiles Identifies Dysregulated Vesicle Trafficking Pathways in Creutzfeldt-Jakob Disease.

Functional genomics applied to the study of RNA expression profiles identified several abnormal molecular processes in experimental prion disease. However, only a few similar studies have been carried out to date in a naturally occurring human prion disease. To better characterize the transcriptional cascades associated with sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, we investigated the global gene expression profile in samples from the frontal cortex of 10 patients with sCJD and 10 non-neurological controls by microarray analysis. The comparison identified 333 highly differentially expressed genes (hDEGs) in sCJD. Functional enrichment Gene Ontology analysis revealed that hDEGs were mainly associated with synaptic transmission, including GABA (q value = 0.049) and glutamate (q value = 0.005) signaling, and the immune/inflammatory response. Furthermore, the analysis of cellular components performed on hDEGs showed a compromised regulation of vesicle-mediated transport with mainly up-regulated genes related to the endosome (q value = 0.01), lysosome (q value = 0.04), and extracellular exosome (q value < 0.01). A targeted analysis of the retromer core component VPS35 (vacuolar protein sorting-associated protein 35) showed a down-regulation of gene expression (p value= 0.006) and reduced brain protein levels (p value= 0.002). Taken together, these results confirm and expand previous microarray expression profile data in sCJD. Most significantly, they also demonstrate the involvement of the endosomal-lysosomal system. Since the latter is a common pathogenic pathway linking together diseases, such as Alzheimer's and Parkinson's, it might be the focus of future studies aimed to identify new therapeutic targets in neurodegenerative diseases.

Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. METHODS: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. RESULTS: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. CONCLUSIONS: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. TRIAL REGISTRATION NUMBER: NCT01725139, pre-clinical.

The 18-kDa mitochondrial translocator protein in gliomas: from the bench to bedside.

The 18-kDa mitochondrial translocator protein (TSPO) is known to be highly expressed in several types of cancer, including gliomas, whereas expression in normal brain is low. TSPO functions in glioma are still incompletely understood. The TSPO can be quantified pre-operatively with molecular imaging making it an ideal candidate for personalized treatment of patient with glioma. Studies have proposed to exploit the TSPO as a transporter of chemotherapics to selectively target tumour cells in the brain. Our studies proved that positron emission tomography (PET)-imaging can contribute to predict progression of patients with glioma and that molecular imaging with TSPO-specific ligands is suitable to stratify patients in view of TSPO-targeted treatment. Finally, we proved that TSPO in gliomas is predominantly expressed by tumour cells.

Both MC1 and MC3 Receptors Provide Protection From Cerebral Ischemia-Reperfusion-Induced Neutrophil Recruitment.

OBJECTIVE: Neutrophil recruitment is a key process in the pathogenesis of stroke, and may provide a valuable therapeutic target. Targeting the melanocortin (MC) receptors has previously shown to inhibit leukocyte recruitment in peripheral inflammation, however, it is not known whether treatments are effective in the unique cerebral microvascular environment. Here, we provide novel research highlighting the effects of the MC peptides on cerebral neutrophil recruitment, demonstrating important yet discrete roles for both MC1 and MC3. APPROACH AND RESULTS: Using intravital microscopy, in 2 distinct murine models of cerebral ischemia-reperfusion (I/R) injury, we have investigated MC control for neutrophil recruitment. After global I/R, pharmacological treatments suppressed pathological neutrophil recruitment. MC1 selective treatment rapidly inhibited neutrophil recruitment while a nonselective MC agonist provided protection even when coadministered with an MC3/4 antagonist, suggesting the importance of early MC1 signaling. However, by 2-hour reperfusion, MC1-mediated effects were reduced, and MC3 anti-inflammatory circuits predominated. Mice bearing a nonfunctional MC1 displayed a transient exacerbation of neutrophil recruitment after global I/R, which diminished by 2 hours. However importantly, enhanced inflammatory responses in both MC1 mutant and MC3 (-/-) mice resulted in increased infarct size and poor functional outcome after focal I/R. Furthermore, we used an in vitro model of leukocyte recruitment to demonstrate these anti-inflammatory actions are also effective in human cells. CONCLUSIONS: These studies reveal for the first time MC control for neutrophil recruitment in the unique pathophysiological context of cerebral I/R, while also demonstrating the potential therapeutic value of targeting multiple MCs in developing effective therapeutics.

Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in neuroinflammation, drugs that mitigate these changes may be neuroprotective when administered to patients with Parkinson's disease.

The 18-kDa mitochondrial translocator protein in human gliomas: an 11C-(R)PK11195 PET imaging and neuropathology study.

UNLABELLED: The 18-kDa mitochondrial translocator protein (TSPO) is upregulated in high-grade astrocytomas and can be imaged by PET using the selective radiotracer (11)C-(R)PK11195. We investigated (11)C-(R)PK11195 binding in human gliomas and its relationship with TSPO expression in tumor tissue and glioma-associated microglia/macrophages (GAMs) within the tumors. METHODS: Twenty-two glioma patients underwent dynamic (11)C-(R)PK11195 PET scans and perfusion MR imaging acquisition. Parametric maps of (11)C-(R)PK11195 binding potential (BPND) were generated. Coregistered MR/PET images were used to guide tumor biopsy. The tumor tissue was quantitatively assessed for TSPO expression and infiltration of GAMs using immunohistochemistry and double immunofluorescence. The imaging and histopathologic parameters were compared among different histotypes and grades and correlated with each other. RESULTS: BPND of (11)C-(R)PK11195 in high-grade gliomas was significantly higher than in low-grade astrocytomas and low-grade oligodendrogliomas. TSPO in gliomas was expressed predominantly by neoplastic cells, and its expression correlated positively with BPND in the tumors. GAMs only partially contributed to the overall TSPO expression within the tumors, and TSPO expression in GAMs did not correlate with tumor BPND. CONCLUSION: PET with (11)C-(R)PK11195 in human gliomas predominantly reflects TSPO expression in tumor cells. It therefore has the potential to effectively stratify patients who are suitable for TSPO-targeted treatment.

Common mechanisms in neurodegeneration and neuroinflammation: a BrainNet Europe gene expression microarray study.

Neurodegenerative diseases of the central nervous system are characterized by pathogenetic cellular and molecular changes in specific areas of the brain that lead to the dysfunction and/or loss of explicit neuronal populations. Despite exhibiting different clinical profiles and selective neuronal loss, common features such as abnormal protein deposition, dysfunctional cellular transport, mitochondrial deficits, glutamate excitotoxicity, iron accumulation and inflammation are observed in many neurodegenerative disorders, suggesting converging pathways of neurodegeneration. We have generated comparative genome-wide gene expression data, using the Illumina HumanRef 8 Beadchip, for Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Parkinson's disease, and schizophrenia using an extensive cohort (n = 113) of well-characterized post-mortem brain tissues. The analysis of whole-genome expression patterns across these major disorders offers an outstanding opportunity not only to look into exclusive disease-specific changes, but more importantly to look for potential common molecular pathogenic mechanisms. Surprisingly, no dysregulated gene that passed our selection criteria was found in common across all six diseases. However, 61 dysregulated genes were shared when comparing five and four diseases. The few genes highlighted by our direct gene comparison analysis hint toward common neuronal homeostatic, survival and synaptic plasticity pathways. In addition, we report changes to several inflammation-related genes in all diseases. This work is supportive of a general role of the innate immune system in the pathogenesis and/or response to neurodegeneration.

Cortical grey matter demyelination can be induced by elevated pro-inflammatory cytokines in the subarachnoid space of MOG-immunized rats.

A substantial proportion of cases with secondary progressive multiple sclerosis have extensive inflammation in the leptomeninges that is associated with increased subpial demyelination, neuronal loss and an exacerbated disease course. However, the mechanisms underlying this extensive subpial pathology are poorly understood. We hypothesize that pro-inflammatory cytokine production within the meninges may be a key to this process. Post-mortem cerebrospinal fluid and dissected cerebral leptomeningeal tissue from patients with multiple sclerosis were used to study the presence of tumour necrosis factor and interferon gamma protein and messenger RNA levels. A novel model of subpial cortical grey matter demyelination was set up in Dark Agouti rats and analysed using quantitative immunohistochemistry. Increased expression of the pro-inflammatory cytokines tumour necrosis factor and interferon gamma was found in the meninges of cases with secondary progressive multiple sclerosis exhibiting tertiary lymphoid-like structures. Injection of tumour necrosis factor and interferon gamma into the subarachnoid space of female Dark Agouti rats pre-immunized with a subclinical dose of myelin oligodendrocyte glycoprotein mimicked the pathology seen in multiple sclerosis, including infiltration of lymphocytes (CD4+ and CD8+ T cells and CD79+ B cells) into the meninges and extensive subpial demyelination. Extensive microglial/macrophage activation was present in a gradient from the pial surface to deeper cortical layers. Demyelination did not occur in control animals immunized with incomplete Freund's adjuvant and injected with cytokines. These results support the hypothesis that pro-inflammatory molecules produced in the meninges play a major role in cortical demyelination in multiple sclerosis, but also emphasize the involvement of an anti-myelin immune response.

Mean Expression of the X-Chromosome is Associated with Neuronal Density.

BACKGROUND: Neurodegenerative diseases are characterized by key features such as loss of neurons, astrocytosis, and microglial activation/proliferation. These changes cause differences in the density of cell types between control and disease subjects, confounding results from gene expression studies. Chromosome X (ChrX) is known to be specifically important in the brain. We hypothesized the existence of a chromosomal signature of gene expression associated with the X-chromosome for neurological conditions not normally associated with that chromosome. The hypothesis was investigated using publicly available microarray datasets from studies on Parkinson's disease, Alzheimer's disease, and Huntington's disease. Data were analyzed using Chromowave, an analytical tool for detecting spatially extended expression changes along chromosomes. To examine associations with neuronal density and astrocytosis, the expression of cell specific reporter genes was extracted. The association between these genes and the expression patterns extracted by Chromowave was then analyzed. Further analyses of the X:Autosome ratios for laser dissected neurons, microglia cultures and whole tissue were performed to detect cell specific differences. RESULTS: We observed an extended pattern of low expression of ChrX consistent in all the neurodegenerative disease brain datasets. There was a strong correlation between mean ChrX expression and the pattern extracted from the autosomal genes representing neurons, but not with mean autosomal expression. No chromosomal patterns associated with the neuron specific genes were found on other chromosomes. The chromosomal expression pattern was not present in datasets from blood cells. The X:Autosome expression ratio was also higher in neuronal cells than in tissues with a mix of cell types. CONCLUSIONS: The results suggest that neurological disorders show as a reduction in mean expression of many genes along ChrX. The most likely explanation for this finding relates to the documented general up-regulation of ChrX in brain tissue which, this work suggests, occurs primarily in neurons. If validated, this cell specific ChrX expression warrants further research as understanding the biological reasons and mechanisms for this expression, may help to elucidate a connection with the development of neurodegenerative disorders.

Increased HLA-E expression in white matter lesions in multiple sclerosis.

The molecular mechanisms underpinning central nervous system damage in multiple sclerosis (MS) are complex and it is widely accepted that there is an autoimmune component. Both adaptive and innate immune effector mechanisms are believed to contribute to tissue disease aetiology. HLA-E is a non-classical MHC class Ib molecule that acts as the ligand for the NKG2A inhibitory receptor present on natural killer (NK) and CD8+ cells. Peptide binding and stabilization of HLA-E is often considered to signal infection or cell stress. Here we examine the up-regulation of HLA-E in MS brain tissue. Expression is significantly increased in white matter lesions in the brain of MS patients compared with white matter of neurologically healthy controls. Furthermore, using quantitative immunohistochemistry and confocal microscopy, we show increased HLA-E protein expression in endothelial cells of active MS lesions. Non-inflammatory chronic lesions express significantly less HLA-E protein, comparable to levels found in white matter from controls. Increased HLA-E protein levels were associated with higher scores of inflammation. These results suggest the potential for an effect in central nervous system pathogenesis from HLA-E modulation in stressed tissue. Co-localization with infiltrating CD8+ cells implicates a possible role for HLA-E-restricted regulatory CD8+ cells, as has been proposed in other autoimmune diseases.

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Inflammatory Pathways in Parkinson's Disease; A BNE Microarray Study.

The aetiology of Parkinson's disease (PD) is yet to be fully understood but it is becoming more and more evident that neuronal cell death may be multifactorial in essence. The main focus of PD research is to better understand substantia nigra homeostasis disruption, particularly in relation to the wide-spread deposition of the aberrant protein α-synuclein. Microarray technology contributed towards PD research with several studies to date and one gene, ALDH1A1 (Aldehyde dehydrogenase 1 family, member A1), consistently reappeared across studies including the present study, highlighting dopamine (DA) metabolism dysfunction resulting in oxidative stress and most probably leading to neuronal cell death. Neuronal cell death leads to increased inflammation through the activation of astrocytes and microglia. Using our dataset, we aimed to isolate some of these pathways so to offer potential novel neuroprotective therapeutic avenues. To that effect our study has focused on the upregulation of P2X7 (purinergic receptor P2X, ligand-gated ion channel, 7) receptor pathway (microglial activation) and on the NOS3 (nitric oxide synthase 3) pathway (angiogenesis). In summary, although the exact initiator of striatal DA neuronal cell death remains to be determined, based on our analysis, this event does not remain without consequence. Extracellular ATP and reactive astrocytes appear to be responsible for the activation of microglia which in turn release proinflammatory cytokines contributing further to the parkinsonian condition. In addition to tackling oxidative stress pathways we also suggest to reduce microglial and endothelial activation to support neuronal outgrowth.

Structural synaptic elements are differentially regulated in superior temporal cortex of schizophrenia patients.

Inaccurate wiring and synaptic pathology appear to be major hallmarks of schizophrenia. A variety of gene products involved in synaptic neurotransmission and receptor signaling are differentially expressed in brains of schizophrenia patients. However, synaptic pathology may also develop by improper expression of intra- and extra-cellular structural elements weakening synaptic stability. Therefore, we have investigated transcription of these elements in the left superior temporal gyrus of 10 schizophrenia patients and 10 healthy controls by genome-wide microarrays (Illumina). Fourteen up-regulated and 22 downregulated genes encoding structural elements were chosen from the lists of differentially regulated genes for further qRT-PCR analysis. Almost all genes confirmed by this method were downregulated. Their gene products belonged to vesicle-associated proteins, that is, synaptotagmin 6 and syntaxin 12, to cytoskeletal proteins, like myosin 6, pleckstrin, or to proteins of the extracellular matrix, such as collagens, or laminin C3. Our results underline the pivotal roles of structural genes that control formation and stabilization of pre- and post-synaptic elements or influence axon guidance in schizophrenia. The glial origin of collagen or laminin highlights the close interrelationship between neurons and glial cells in establishment and maintenance of synaptic strength and plasticity. It is hypothesized that abnormal expression of these and related genes has a major impact on the pathophysiology of schizophrenia.

Innate immunity in multiple sclerosis white matter lesions: expression of natural cytotoxicity triggering receptor 1 (NCR1).

BACKGROUND: Pathogenic or regulatory effects of natural killer (NK) cells are implicated in many autoimmune diseases, but evidence in multiple sclerosis (MS) and its murine models remains equivocal. In an effort to illuminate this, we have here analysed expression of the prototypic NK cell marker, NCR1 (natural cytotoxicity triggering receptor; NKp46; CD335), an activating receptor expressed by virtually all NK cells and therefore considered a pan-marker for NK cells. The only definitive ligand of NCR1 is influenza haemagglutinin, though there are believed to be others. In this study, we investigated whether there were differences in NCR1+ cells in the peripheral blood of MS patients and whether NCR1+ cells are present in white matter lesions. RESULTS: We first investigated the expression of NCR1 on peripheral blood mononuclear cells and found no significant difference between healthy controls and MS patients. We then investigated mRNA levels in central nervous system (CNS) tissue from MS patients: NCR1 transcripts were increased more than 5 times in active disease lesions. However when we performed immunohistochemical staining of this tissue, few NCR1+ NK cells were identified. Rather, the major part of NCR1 expression was localised to astrocytes, and was considerably more pronounced in MS patients than controls. In order to further validate de novo expression of NCR1 in astrocytes, we used an in vitro staining of the human astrocytoma U251 cell line grown to model whether cell stress could be associated with expression of NCR1. We found up-regulation of NCR1 expression in U251 cells at both the mRNA and protein levels. CONCLUSIONS: The data presented here show very limited expression of NCR1+ NK cells in MS lesions, the majority of NCR1 expression being accounted for by expression on astrocytes. This is compatible with a role of this cell-type and NCR1 ligand/receptor interactions in the innate immune response in the CNS in MS patients. This is the first report of NCR1 expression on astrocytes in MS tissue: it will now be important to unravel the nature of cellular interactions and signalling mediated through innate receptor expression on astrocytes.