Peripheral reduction of ß-amyloid is sufficient to reduce brain ß-amyloid: implications for Alzheimer's disease.

Three loci that modify ß-amyloid (Aß) accumulation and deposition in the brains of a mouse model of Alzheimer's disease have been previously described. One encompasses the Psen2 gene encoding presenilin 2, a component of the γ-secretase activity responsible for generating Aß by proteolysis. We show that the activity of mouse Psen2, as measured by levels of mRNA accumulation, unexpectedly is heritable in the liver but not the brain, suggesting liver as the origin of brain Aß deposits. Administration of STI571, a cancer therapeutic that does not cross the blood-brain barrier, reduced accumulation of Aß in both the blood and the brain, confirming brain Aß's peripheral origin and suggesting that STI571 and related compounds might have therapeutic/prophylactic value in human Alzheimer's disease. The genes Cib1 and Zfhx1b reside within the other modifier loci and also exhibit heritable expression in the liver, suggesting that they too contribute to Aß accumulation.

Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes.

A central tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.

Regulation of alpha-synuclein expression in limbic and motor brain regions of morphine-treated mice.

Chronic exposure to opiates produces dependence and addiction, which may result from neuroadaptations in the dopaminergic reward pathway and its target brain regions. The neuronal protein alpha-synuclein has been implicated in neuronal plasticity and proposed to serve as a negative regulator of dopamine neurotransmission. Thus, alpha-synuclein could mediate some effects of opiates in the brain. The present study investigated the influence of acute and chronic morphine administration on alpha-synuclein mRNA and protein expression in the brains of mice. Downregulation of alpha-synuclein mRNA was observed in the basolateral amygdala, dorsal striatum, nucleus accumbens, and ventral tegmental area of mice withdrawn from chronic morphine treatment. The changes were the most pronounced after longer periods of withdrawal (48 h). In contrast, levels of alpha-synuclein protein, as assessed by Western blotting, were significantly increased in the amygdala and striatum/accumbens (but not in the mesencephalon) of morphine-withdrawn mice. In both brain regions, levels of alpha-synuclein were elevated for as long as 2 weeks after treatment cessation. Because alpha-synuclein is a presynaptic protein, the detected opposite changes in its mRNA and protein levels are likely to take place in different populations of projection neurons whose somata are in different brain areas. Axonal localization of alpha-synuclein was confirmed by immunofluorescent labeling. An attempt to identify postsynaptic neurons innervated by alpha-synuclein-containing axon terminals revealed their selective apposition to calbindin D28K-negative projection neurons in the basolateral amygdala. The observed changes in alpha-synuclein levels are discussed in connection with their putative role in mediating suppression of dopaminergic neurotransmission during opiate withdrawal.

Altered gut microbial energy and metabolism in children with non-alcoholic fatty liver disease.

Obesity is becoming the new pediatric epidemic. Non-alcoholic fatty liver disease (NAFLD) is frequently associated with obesity and has become the most common cause of pediatric liver disease. The gut microbiome is the major metabolic organ and determines how calories are processed, serving as a caloric gate and contributing towards the pathogenesis of NAFLD. The goal of this study is to examine gut microbial profiles in children with NAFLD using phylogenetic, metabolomic, metagenomic and proteomic approaches. Fecal samples were obtained from obese children with or without NAFLD and healthy lean children. Stool specimens were subjected to 16S rRNA gene microarray, shotgun sequencing, mass spectroscopy for proteomics and NMR spectroscopy for metabolite analysis. Children with NAFLD had more abundant Gammaproteobacteria and Prevotella and significantly higher levels of ethanol, with differential effects on short chain fatty acids. This group also had increased genomic and protein abundance for energy production with a reduction in carbohydrate and amino acid metabolism and urea cycle and urea transport systems. The metaproteome and metagenome showed similar findings. The gut microbiome in pediatric NAFLD is distinct from lean healthy children with more alcohol production and pathways allocated to energy metabolism over carbohydrate and amino acid metabolism, which would contribute to development of disease.

Clinical pertinence metric enables hypothesis-independent genome-phenome analysis for neurologic diagnosis.

We describe an "integrated genome-phenome analysis" that combines both genomic sequence data and clinical information for genomic diagnosis. It is novel in that it uses robust diagnostic decision support and combines the clinical differential diagnosis and the genomic variants using a "pertinence" metric. This allows the analysis to be hypothesis-independent, not requiring assumptions about mode of inheritance, number of genes involved, or which clinical findings are most relevant. Using 20 genomic trios with neurologic disease, we find that pertinence scores averaging 99.9% identify the causative variant under conditions in which a genomic trio is analyzed and family-aware variant calling is done. The analysis takes seconds, and pertinence scores can be improved by clinicians adding more findings. The core conclusion is that automated genome-phenome analysis can be accurate, rapid, and efficient. We also conclude that an automated process offers a methodology for quality improvement of many components of genomic analysis.

Catching target receptors for drug and vaccine delivery using TOGA gene expression profiling.

Drug delivery technologies are commonly directed towards formulations to control the delivery of therapeutic compounds. However, many processes in the human body have evolved to regulate the transport of various molecules, cells, or particles across epithelial barriers. To take advantage of this biology, we used TOGA gene expression profiling to identify receptor or transporter molecules to target delivery vehicles for transport across an epithelial barrier. In the case of intestinal epithelium, we sought molecules associated with the transport of particles by Peyer's patch M cells. We have identified genes specific to Peyer's patch epithelium, some of which appear to be M cell specific. Discoveries made by this process will provide targets for development of new vaccines, but also provide new insights into the biology of transepithelial transport. The power of this gene profiling approach also suggests application to other systems, such as the response to metabolic changes or drug treatments.

Open system gene expression profiling and identification of novel genes for targeted vaccine delivery.

Ongoing development of vaccines is limited by the techniques used to identify routes to immunization. Using open system gene expression profiling, researchers can now directly identify receptors used by M-cells in the transport of antigens to the mucosal immune system. These receptors will be ideal for targeted delivery of new synthetic vaccines. In addition, gene expression profiling will provide important new information on the biology of the M-cell and mucosal immune mechanisms.

Joint variant and de novo mutation identification on pedigrees from high-throughput sequencing data.

The analysis of whole-genome or exome sequencing data from trios and pedigrees has been successfully applied to the identification of disease-causing mutations. However, most methods used to identify and genotype genetic variants from next-generation sequencing data ignore the relationships between samples, resulting in significant Mendelian errors, false positives and negatives. Here we present a Bayesian network framework that jointly analyzes data from all members of a pedigree simultaneously using Mendelian segregation priors, yet providing the ability to detect de novo mutations in offspring, and is scalable to large pedigrees. We evaluated our method by simulations and analysis of whole-genome sequencing (WGS) data from a 17-individual, 3-generation CEPH pedigree sequenced to 50× average depth. Compared with singleton calling, our family caller produced more high-quality variants and eliminated spurious calls as judged by common quality metrics such as Ti/Tv, Het/Hom ratios, and dbSNP/SNP array data concordance, and by comparing to ground truth variant sets available for this sample. We identify all previously validated de novo mutations in NA12878, concurrent with a 7× precision improvement. Our results show that our method is scalable to large genomics and human disease studies.

Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing.

Distinguishing single-nucleotide variants (SNVs) from errors in whole-genome sequences remains challenging. Here we describe a set of filters, together with a freely accessible software tool, that selectively reduce error rates and thereby facilitate variant detection in data from two short-read sequencing technologies, Complete Genomics and Illumina. By sequencing the nearly identical genomes from monozygotic twins and considering shared SNVs as 'true variants' and discordant SNVs as 'errors', we optimized thresholds for 12 individual filters and assessed which of the 1,048 filter combinations were effective in terms of sensitivity and specificity. Cumulative application of all effective filters reduced the error rate by 290-fold, facilitating the identification of genetic differences between monozygotic twins. We also applied an adapted, less stringent set of filters to reliably identify somatic mutations in a highly rearranged tumor and to identify variants in the NA19240 HapMap genome relative to a reference set of SNVs.

New prospects and strategies for drug target discovery in neurodegenerative disorders.

The future of neurodegenerative therapeutics development depends upon effective disease modification strategies centered on carefully investigated targets. Pharmaceutical research endeavors that probe for a much deeper understanding of disease pathogenesis, and explain how adaptive or compensatory mechanisms might be engaged to delay disease onset or progression, will produce the needed breakthroughs. Below, we discuss the prospects for new targets emerging out of the study of brain disease genes and their associated pathogenic pathways. We describe a general experimental paradigm that we are employing across several mouse models of neurodegenerative disease to elucidate molecular determinants of selective neuronal vulnerability. We outline key elements of our target discovery program and provide examples of how we integrate genomic technologies, neuroanatomical methods, and mouse genetics in the search for neurodegenerative disease targets.

Identification of complement 5a-like receptor (C5L2) from astrocytes: characterization of anti-inflammatory properties.

Brain inflammation is regulated by endogenous substances, including neurotransmitters such as noradrenaline (NA), which can increase anti-inflammatory genes. To identify NA-regulated, anti-inflammatory genes, we used TOGA (total gene expression analysis) to screen rat astrocyte-derived RNA. NA-inducible cDNA clone DST11 encodes an isoform of the complement C5a receptor (C5aR), with 39% identity at the amino acid level to the rat C5aR, and 56% identity to a recently described human C5aR variant termed C5L2 (complement 5a-like receptor). Quantitative PCR confirmed that in astrocytes, DST11 mRNA expression is increased by NA, whereas in vivo depletion of cortical NA reduced DST11 levels. Western blot analysis demonstrated basal and NA-induced expression of DST11 as a 45 kDa protein in primary astrocytes cultures. Immunocytochemical staining of adult rat brain revealed DST11-immunoreactivity throughout brain, co-localized to neurons and astrocytes. In astrocytes, induction of nitric oxide synthase type 2 was increased by treatment with antisense oligonucleotides to DST11. Reducing DST11 expression also increased nuclear factor kappaB reporter gene, and decreased cAMP response element reporter gene activation. These results demonstrate that DST11 is a C5aR isoform expressed by glia and neurons, which is regulated by NA, and exerts anti-inflammatory functions. Changes in DST11 levels in diseased brain could therefore contribute to the progression of inflammatory damage.

Aberrant recombination involving the granzyme locus occurs in Atm-/- T-cell lymphomas.

Ataxia telangiectasia (A-T) is an autosomal recessive disease caused by loss of function of the serine/threonine protein kinase ATM (ataxia telangiectasia mutated). A-T patients have a 250-700-fold increased risk of developing lymphomas and leukemias which are typically highly invasive and proliferative. In addition, a subset of adult acute lymphoblastic leukemias and aggressive B-cell chronic lymphocytic leukemias that occur in the general population show loss of heterozygosity for ATM. To define the specific role of ATM in lymphomagenesis, we studied T-cell lymphomas isolated from mice with mutations in ATM and/or p53 using cytogenetic analysis and mRNA transcriptional profiling. The analyses identified genes misregulated as a consequence of the amplifications, deletions and translocation events arising as a result of ATM loss. A specific recurrent disruption of the granzyme gene family locus was identified resulting in an aberrant granzyme B/C fusion product. The combined application of cytogenetic and gene expression approaches identified specific loci and genes that define the pathway of initiation and progression of lymphoreticular malignancies in the absence of ATM.

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia.

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.

Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma inhibit hepatitis B virus (HBV) replication noncytopathically in the livers of HBV transgenic mice and in hepatocyte cell lines derived from these mice. The present study was designed to identify transcriptionally controlled hepatocellular genes that are tightly associated with the inhibition of HBV replication and that might, therefore, mediate the antiviral effect of these cytokines. Twenty-nine genes were identified, many of which have known or potential antiviral activity. Notably, multiple components of the immunoproteasome and ubiquitin-like proteins were strongly induced by both IFN-alpha/beta and IFN-gamma, as were a number of GTP-binding proteins, including GTPases with known antiviral activity, chemokines, signaling molecules, and miscellaneous genes associated with antigen processing, DNA-binding, or cochaperone activity and several expressed sequence tags. The results suggest that one or more members of this relatively small subset of genes may mediate the antiviral effect of IFN-alpha/beta and IFN-gamma against HBV. We have already exploited this information by demonstrating that the antiviral activity of IFN-alpha/beta and IFN-gamma is proteasome dependent.