Winnie S. Liang

GAB2 Alleles Modify Alzheimer's Risk in APOE ε4 Carriers

The apolipoprotein E (APOE) epsilon4 allele is the best established genetic risk factor for late-onset Alzheimers disease (LOAD). We conducted genome-wide surveys of 502,627 single-nucleotide polymorphisms (SNPs) to characterize and confirm other LOAD susceptibility genes. In epsilon4 carriers from neuropathologically verified discovery, neuropathologically verified replication, and clinically characterized replication cohorts of 1411 cases and controls, LOAD was associated with six SNPs from the GRB-associated binding protein 2 (GAB2) gene and a common haplotype encompassing the entire GAB2 gene. SNP rs2373115 (p = 9 x 10(-11)) was associated with an odds ratio of 4.06 (confidence interval 2.81-14.69), which interacts with APOE epsilon4 to further modify risk. GAB2 was overexpressed in pathologically vulnerable neurons; the Gab2 protein was detected in neurons, tangle-bearing neurons, and dystrophic neuritis; and interference with GAB2 gene expression increased tau phosphorylation. Our findings suggest that GAB2 modifies LOAD risk in APOE epsilon4 carriers and influences Alzheimers neuropathology.

Alzheimer's disease is associated with reduced expression of energy metabolism genes in posterior cingulate neurons

Alzheimers disease (AD) is associated with regional reductions in fluorodeoxyglucose positron emission tomography (FDG PET) measurements of the cerebral metabolic rate for glucose, which may begin long before the onset of histopathological or clinical features, especially in carriers of a common AD susceptibility gene. Molecular evaluation of cells from metabolically affected brain regions could provide new information about the pathogenesis of AD and new targets at which to aim disease-slowing and prevention therapies. Data from a genome-wide transcriptomic study were used to compare the expression of 80 metabolically relevant nuclear genes from laser-capture microdissected non-tangle-bearing neurons from autopsy brains of AD cases and normal controls in posterior cingulate cortex, which is metabolically affected in the earliest stages; other brain regions metabolically affected in PET studies of AD or normal aging; and visual cortex, which is relatively spared. Compared with controls, AD cases had significantly lower expression of 70% of the nuclear genes encoding subunits of the mitochondrial electron transport chain in posterior cingulate cortex, 65% of those in the middle temporal gyrus, 61% of those in hippocampal CA1, 23% of those in entorhinal cortex, 16% of those in visual cortex, and 5% of those in the superior frontal gyrus. Western blots confirmed underexpression of those complex I–V subunits assessed at the protein level. Cerebral metabolic rate for glucose abnormalities in FDG PET studies of AD may be associated with reduced neuronal expression of nuclear genes encoding subunits of the mitochondrial electron transport chain.

Altered neuronal gene expression in brain regions differentially affected by Alzheimer's disease: a reference data set

Alzheimers Disease (AD) is the most widespread form of dementia during the later stages of life. If improved therapeutics are not developed, the prevalence of AD will drastically increase in the coming years as the worlds population ages. By identifying differences in neuronal gene expression profiles between healthy elderly persons and individuals diagnosed with AD, we may be able to better understand the molecular mechanisms that drive AD pathogenesis, including the formation of amyloid plaques and neurofibrillary tangles. In this study, we expression profiled histopathologically normal cortical neurons collected with laser capture microdissection (LCM) from six anatomically and functionally discrete postmortem brain regions in 34 AD-afflicted individuals, using Affymetrix Human Genome U133 Plus 2.0 microarrays. These regions include the entorhinal cortex, hippocampus, middle temporal gyrus, posterior cingulate cortex, superior frontal gyrus, and primary visual cortex. This study is predicated on previous parallel research on the postmortem brains of the same six regions in 14 healthy elderly individuals, for which LCM neurons were similarly processed for expression analysis. We identified significant regional differential expression in AD brains compared with control brains including expression changes of genes previously implicated in AD pathogenesis, particularly with regard to tangle and plaque formation. Pinpointing the expression of factors that may play a role in AD pathogenesis provides a foundation for future identification of new targets for improved AD therapeutics. We provide this carefully phenotyped, laser capture microdissected intraindividual brain region expression data set to the community as a public resource.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

Evidence for an association between KIBRA and late-onset Alzheimer's disease

We recently reported evidence for an association between the individual variation in normal human episodic memory and a common variant of the KIBRA gene, KIBRA rs17070145 (T-allele). Since memory impairment is a cardinal clinical feature of Alzheimers disease (AD), we investigated the possibility of an association between the KIBRA gene and AD using data from neuronal gene expression, brain imaging studies, and genetic association tests. KIBRA was significantly over-expressed and three of its four known binding partners under-expressed in AD-affected hippocampal, posterior cingulate and temporal cortex regions (P<0.010, corrected) in a study of laser-capture microdissected neurons. Using positron emission tomography in a cohort of cognitively normal, late-middle-aged persons genotyped for KIBRA rs17070145, KIBRA T non-carriers exhibited lower glucose metabolism than did carriers in posterior cingulate and precuneus brain regions (P<0.001, uncorrected). Lastly, non-carriers of the KIBRA rs17070145 T-allele had increased risk of late-onset AD in an association study of 702 neuropathologically verified expired subjects (P=0.034; OR=1.29) and in a combined analysis of 1026 additional living and expired subjects (P=0.039; OR=1.26). Our findings suggest that KIBRA is associated with both individual variation in normal episodic memory and predisposition to AD.

A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells

IntroductionCyclo-oxygenase (COX)-2 expression correlates directly with highly aggressive and metastatic breast cancer, but the mechanism underlying this correlation remains obscure. We hypothesized that invasive human breast cancer cells that over-express COX-2 have the unique ability to differentiate into extracellular-matrix-rich vascular channels, also known as vasculogenic mimicry. Vascular channels have been associated with angiogenesis without involvement of endothelial cells, and may serve as another mechanism by which tumor cells obtain nutrients to survive, especially in less vascularized regions of the tumor.MethodsTo determine whether COX-2 regulates vascular channel formation, we assessed whether treatment with celecoxib (a selective COX-2 inhibitor) or silencing COX-2 synthesis by siRNA inhibits vascular channel formation by breast cancer cell lines. Cell lines were selected based on their invasive potential and COX-2 expression. Additionally, gene expression analysis was performed to identify candidate genes involved in COX-2-induced vascular channel formation. Finally, vascular channels were analyzed in surgically resected human breast cancer specimens that expressed varying levels of COX-2.ResultsWe found that invasive human breast cancer cells that over-express COX-2 develop vascular channels when plated on three-dimensional matigel cultures, whereas non-invasive cell lines that express low levels of COX-2 did not develop such channels. Similarly, we identified vascular channels in high-grade invasive ductal carcinoma of the breast over-expressing COX-2, but not in low-grade breast tumors. Vascular channel formation was significantly suppressed when cells were treated with celecoxib or COX-2 siRNA. Inhibition of channel formation was abrogated by addition of exogenous prostaglandin E2. In vitro results were corroborated in vivo in tumor-bearing mice treated with celecoxib. Using gene expression profiling, we identified several genes in the angiogenic and survival pathways that are engaged in vascular channel formation.ConclusionAntivascular therapies targeting tumor cell vasculogenic mimicry may be an effective approach to the treatment of patients with highly metastatic breast cancer.

Alzheimer's disease is associated with altered expression of genes involved in immune response and mitochondrial processes in astrocytes

Alzheimers disease (AD) is characterized by deficits in cerebral metabolic rates of glucose in the posterior cingulate (PC) and precuneus in AD subjects, and in APOEε4 carriers, decades before the onset of measureable cognitive deficits. However, the cellular and molecular basis of this phenotype remains to be clarified. Given the roles of astrocytes in energy storage and brain immunity, we sought to characterize the transcriptome of AD PC astrocytes. Cells were laser capture microdissected from AD (n = 10) and healthy elderly control (n = 10) subjects for RNA sequencing. We generated >5.22 billion reads and compared sequencing data between controls and AD patients. We identified differentially expressed mitochondria-related genes including TRMT61B, FASTKD2, and NDUFA4L2, and using pathway and weighted gene coexpression analyses, we identified differentially expressed immune response genes. A number of these genes, including CLU, C3, and CD74, have been implicated in beta amyloid generation or clearance. These data provide key insights into astrocyte-specific contributions to AD, and we present this data set as a publicly available resource.

Neuronal gene expression in non-demented individuals with intermediate Alzheimer's Disease neuropathology.

While the clinical and neuropathological characterization of Alzheimers Disease (AD) is well defined, our understanding of the progression of pathologic mechanisms in AD remains unclear. Post-mortem brains from individuals who did not fulfill clinical criteria for AD may still demonstrate measurable levels of AD pathologies to suggest that they may have presented with clinical symptoms had they lived longer or are able to stave off disease progression. Comparison between such individuals and those clinically diagnosed and pathologically confirmed to have AD will be key in delineating AD pathogenesis and neuroprotection. In this study, we expression profiled laser capture microdissected non-tangle bearing neurons in 6 post-mortem brain regions that are differentially affected in the AD brain from 10 non-demented individuals demonstrating intermediate AD neuropathologies (NDAD; Braak stage of II through IV and CERAD rating of moderate to frequent) and evaluated this data against that from individuals who have been diagnosed with late onset AD as well as healthy elderly controls. We identified common statistically significant expression changes in both NDAD and AD brains that may establish a degenerative link between the two cohorts, in addition to NDAD specific transcriptomic changes. These findings pinpoint novel targets for developing earlier diagnostics and preventative therapies for AD prior to diagnosis of probable AD. We also provide this high-quality, low post-mortem interval (PMI), cell-specific, and region-specific NDAD/AD reference data set to the community as a public resource.

Tonic Premarin dose-dependently enhances memory, affects neurotrophin protein levels and alters gene expression in middle-aged rats

Premarin™ is the most commonly prescribed estrogenic component of hormone therapy, given since 1942. The current study is the first examining cognitive effects of tonic Premarin treatment in an animal model. Middle-aged ovariectomized (Ovx) rats received vehicle or one of three doses of Premarin (12, 24 or 36μg daily). Rats were tested on a spatial working and reference memory maze battery. Both medium- and high-dose Premarin enhanced memory retention, while low-dose Premarin impaired learning and memory retention. Correlations with serum hormone levels showed that as the ratio of estrone:17β-estradiol increased, animals tended to show better working memory performance. Taken together with the dissociation of dose-specific estrogenic profiles, results suggest that higher levels of estrone, in the presence of 17β-estradiol concentrations higher than that of Ovx levels, may be beneficial for memory. Moreover, Premarin exerted dose and brain-region specific effects on BDNF and NGF protein levels, with most marked changes in cingulate and perirhinal cortices. Hippocampal gene expression profiling demonstrated significant Premarin-induced transcriptional changes in genes linked to plasticity and cognition. These findings indicate that Premarin can impact memory and the brain, and that dosing should be recognized as a clinically relevant factor possibly affecting the direction and efficacy of cognitive outcome.

Comparative RNA-Seq and Microarray Analysis of Gene Expression Changes in B-Cell Lymphomas of Canis familiaris

Comparative oncology is a developing research discipline that is being used to assist our understanding of human neoplastic diseases. Companion canines are a preferred animal oncology model due to spontaneous tumor development and similarity to human disease at the pathophysiological level. We use a paired RNA sequencing (RNA-Seq)/microarray analysis of a set of four normal canine lymph nodes and ten canine lymphoma fine needle aspirates to identify technical biases and variation between the technologies and convergence on biological disease pathways. Surrogate Variable Analysis (SVA) provides a formal multivariate analysis of the combined RNA-Seq/microarray data set. Applying SVA to the data allows us to decompose variation into contributions associated with transcript abundance, differences between the technology, and latent variation within each technology. A substantial and highly statistically significant component of the variation reflects transcript abundance, and RNA-Seq appeared more sensitive for detection of transcripts expressed at low levels. Latent random variation among RNA-Seq samples is also distinct in character from that impacting microarray samples. In particular, we observed variation between RNA-Seq samples that reflects transcript GC content. Platform-independent variable decomposition without a priori knowledge of the sources of variation using SVA represents a generalizable method for accomplishing cross-platform data analysis. We identified genes differentially expressed between normal lymph nodes of disease free dogs and a subset of the diseased dogs diagnosed with B-cell lymphoma using each technology. There is statistically significant overlap between the RNA-Seq and microarray sets of differentially expressed genes. Analysis of overlapping genes in the context of biological systems suggests elevated expression and activity of PI3K signaling in B-cell lymphoma biopsies compared with normal biopsies, consistent with literature describing successful use of drugs targeting this pathway in lymphomas.