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1.  MTOR regulates the pro-tumorigenic senescence-associated secretory phenotype by promoting IL1A translation 
Nature cell biology  2015;17(8):1049-1061.
The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.
doi:10.1038/ncb3195
PMCID: PMC4691706  PMID: 26147250
2.  Reassessment of Risk Genotypes (GRN, TMEM106B, and ABCC9 Variants) Associated with Hippocampal Sclerosis of Aging Pathology 
Hippocampal sclerosis of aging (HS-Aging) is a common, high morbidity-associated neurodegenerative condition in elderly persons. To understand risk factors for HS-Aging, we analyzed data from the Alzheimer’s Disease Genetics Consortium and correlated the data with clinical and pathologic information from the National Alzheimer’s Coordinating Center database. Overall, 268 research volunteers with HS-Aging and 2957 controls were included; detailed neuropathologic data were available for all. The study focused on single nucleotide polymorphisms previously associated with HS-Aging risk: rs5848 (GRN), rs1990622 (TMEM106B), and rs704180 (ABCC9). Analyses of a subsample that were not previously evaluated (51 HS-Aging cases and 561 controls) replicated the associations of previously identified HS-Aging risk alleles. To test for evidence of gene-gene interactions and genotype-phenotype relationships, pooled data were analyzed. The risk for HS-Aging diagnosis associated with these genetic polymorphisms was not secondary to an association with either Alzheimer disease or dementia with Lewy bodies neuropathologic changes. The presence of multiple risk genotypes was associated with a trend for additive risk for HS-Aging pathology. We conclude that multiple genes play important roles in HS-Aging, which is a distinctive neurodegenerative disease of aging.
doi:10.1097/NEN.0000000000000151
PMCID: PMC4270894  PMID: 25470345
Alzheimer disease; Hippocampal sclerosis; KATP; SUR2; Genome wide association study (GWAS); Progranulin; Frontotemporal lobar degeneration (FTLD)
3.  A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer 
The Prostate  2015;75(11):1206-1215.
BACKGROUND
Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study.
METHODS
We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up.
RESULTS
In 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P < 0.0001), seminal vesicle invasion (P = 0.0008), extracapsular extension (P < 0.0001), and Gleason score (P = 0.0002). In logistic regression analysis of clinical and pathological variables, PTEN deletion was significantly associated with extracapsular extension, seminal vesicle involvement, and higher Gleason score. In the 406 patients in which clinical information was available, PTEN homozygous (P = 0.009) deletion was associated with worse post-operative recurrence-free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P < 0.001), and pathologic stage (P = 0.03).
CONCLUSION
PTEN status assessed by FISH is an independent predictor for recurrence-free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic. Prostate 75: 1206–1215, 2015. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.
doi:10.1002/pros.23003
PMCID: PMC4475421  PMID: 25939393
active surveillance; Gleason score; biomarker; PI3K/PTEN/Akt pathway; fluorescence in situ hybridization; tissue array analysis
4.  Primary age-related tauopathy (PART): a common pathology associated with human aging 
Acta neuropathologica  2014;128(6):755-766.
We recommend a new term, “primary age-related tauopathy” (PART), to describe a pathology that is commonly observed in the brains of aged individuals. Many autopsy studies have reported brains with neurofibrillary tangles (NFT) that are indistinguishable from those of Alzheimer's disease (AD), in the absence of amyloid (Aβ) plaques. For these “NFT+/Aβ−” brains, for which formal criteria for AD neuropathologic changes are not met, the NFT are mostly restricted to structures in the medial temporal lobe, basal forebrain, brainstem, and olfactory areas (bulb and cortex). Symptoms in persons with PART usually range from normal to amnestic cognitive changes, with only a minority exhibiting profound impairment. Because cognitive impairment is often mild, existing clinicopathologic designations, such as “tangle-only dementia” and “tangle-predominant senile dementia”, are imprecise and not appropriate for most subjects. PART is almost universally detectable at autopsy among elderly individuals, yet this pathological process cannot be specifically identified pre-mortem at the present time. Improved biomarkers and tau imaging may enable diagnosis of PART in clinical settings in the future. Indeed, recent studies have identified a common biomarker profile consisting of temporal lobe atrophy and tauopathy without evidence of Aβ accumulation. For both researchers and clinicians, a revised nomenclature will raise awareness of this extremely common pathologic change while providing a conceptual foundation for future studies. Prior reports that have elucidated features of the pathologic entity we refer to as PART are discussed, and working neuropathological diagnostic criteria are proposed.
doi:10.1007/s00401-014-1349-0
PMCID: PMC4257842  PMID: 25348064
TPSD; TOD; Braak; Neuropathology; consensus
5.  Strategy and rationale for urine collection protocols employed in the NEPTUNE study 
BMC Nephrology  2015;16:190.
Background
Glomerular diseases are potentially fatal, requiring aggressive interventions and close monitoring. Urine is a readily-accessible body fluid enriched in molecular signatures from the kidney and therefore particularly suited for routine clinical analysis as well as development of non-invasive biomarkers for glomerular diseases.
Methods
The Nephrotic Syndrome Study Network (NEPTUNE; ClinicalTrials.gov Identifier NCT01209000) is a North American multicenter collaborative consortium established to develop a translational research infrastructure for nephrotic syndrome. This includes standardized urine collections across all participating centers for the purpose of discovering non-invasive biomarkers for patients with nephrotic syndrome due to minimal change disease, focal segmental glomerulosclerosis, and membranous nephropathy. Here we describe the organization and methods of urine procurement and banking procedures in NEPTUNE.
Results
We discuss the rationale for urine collection and storage conditions, and demonstrate the performance of three experimental analytes (neutrophil gelatinase-associated lipocalin [NGAL], retinol binding globulin, and alpha-1 microglobulin) under these conditions with and without urine preservatives (thymol, toluene, and boric acid). We also demonstrate the quality of RNA and protein collected from the urine cellular pellet and exosomes.
Conclusions
The urine collection protocol in NEPTUNE allows robust detection of a wide range of proteins and RNAs from urine supernatant and pellets collected longitudinally from each patient over 5 years. Combined with the detailed clinical and histopathologic data, this provides a unique resource for exploration and validation of new or accepted markers of glomerular diseases.
Trial registration
ClinicalTrials.gov Identifier NCT01209000
Electronic supplementary material
The online version of this article (doi:10.1186/s12882-015-0185-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12882-015-0185-3
PMCID: PMC4650313  PMID: 26577187
Exosome; Urinalysis; Urine specimen collection
6.  NEDD4-mediated HSF1 degradation underlies α-synucleinopathy 
Human Molecular Genetics  2015;25(2):211-222.
Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin–proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.
doi:10.1093/hmg/ddv445
PMCID: PMC4706110  PMID: 26503960
7.  Self-reported memory complaints 
Neurology  2014;83(15):1359-1365.
Objective:
We assessed salience of subjective memory complaints (SMCs) by older individuals as a predictor of subsequent cognitive impairment while accounting for risk factors and eventual neuropathologies.
Methods:
Subjects (n = 531) enrolled while cognitively intact at the University of Kentucky were asked annually if they perceived changes in memory since their last visit. A multistate model estimated when transition to impairment occurred while adjusting for intervening death. Risk factors affecting the timing and probability of an impairment were identified. The association between SMCs and Alzheimer-type neuropathology was assessed from autopsies (n = 243).
Results:
SMCs were reported by more than half (55.7%) of the cohort, and were associated with increased risk of impairment (unadjusted odds ratio = 2.8, p < 0.0001). Mild cognitive impairment (dementia) occurred 9.2 (12.1) years after SMC. Multistate modeling showed that SMC reporters with an APOE ε4 allele had double the odds of impairment (adjusted odds ratio = 2.2, p = 0.036). SMC smokers took less time to transition to mild cognitive impairment, while SMC hormone-replaced women took longer to transition directly to dementia. Among participants (n = 176) who died without a diagnosed clinical impairment, SMCs were associated with elevated neuritic amyloid plaques in the neocortex and medial temporal lobe.
Conclusion:
SMC reporters are at a higher risk of future cognitive impairment and have higher levels of Alzheimer-type brain pathology even when impairment does not occur. As potential harbingers of future cognitive decline, physicians should query and monitor SMCs from their older patients.
doi:10.1212/WNL.0000000000000856
PMCID: PMC4189103  PMID: 25253756
8.  White matter integrity is associated with CSF markers of AD in normal adults 
Neurobiology of aging  2014;35(10):2263-2271.
We explored whether white matter (WM) integrity in cognitively normal (CN) older adults is associated with cerebrospinal fluid (CSF) markers of Alzheimer’s disease (AD) pathology. Twenty CN older adults underwent lumbar puncture and magnetic resonance imaging within a few days of each other. Analysis of diffusion tensor imaging data involved a priori region of interest (ROI) and voxelwise approaches. The ROI results revealed a positive correlation between CSF measures of amyloid-beta (Aβ42 and Aβ42/p-Tau181) and WM integrity in the fornix, a relationship which persisted after controlling for hippocampal volume and fornix volume. Lower WM integrity in the same portion of the fornix was also associated with reduced performance on the Digit Symbol test. Subsequent exploratory voxelwise analyses indicated a positive correlation between CSF Aβ42/p-Tau181 and WM integrity in bilateral portions of the fornix, superior longitudinal fasciculus, inferior fronto-occipital fasciculus, and in the corpus callosum and left inferior longitudinal fasciculus. Our results link lower WM microstructural integrity in CN older adults with CSF biomarkers of AD and suggest that this association in the fornix may be independent of volumetric measures.
doi:10.1016/j.neurobiolaging.2014.04.030
PMCID: PMC4087077  PMID: 24866404
Normal older adults; CSF; white matter; DTI; preclinical AD
9.  Spheroid culture of LuCaP 147 as an authentic preclinical model of prostate cancer subtype with SPOP mutation and hypermutator phenotype 
Cancer letters  2014;351(2):272-280.
LuCaP serially transplantable xenografts are valuable preclinical models of locally advanced or metastatic prostate cancer. For the first time, we recently succeeded in establishing and serially passaging spheroid cultures of several LuCaP xenografts. Here, we characterized in depth the molecular and cellular phenotype of LuCaP 147 cultures and found faithful retention of the characteristics of the original xenograft, including immunophenotype, genetic fidelity, gene expression profile and responsiveness to androgen. Furthermore, we demonstrated capabilities for high-throughput drug screening and that anti-cancer agents induced cell cycle arrest and apoptosis in spheroid cultures. Finally, we showed that cells formed tumors when re-introduced into mice, providing an authentic in vitro - in vivo preclinical model of a subtype of prostate cancer with a hypermutator phenotype and an SPOP mutation.
doi:10.1016/j.canlet.2014.06.014
PMCID: PMC4112013  PMID: 24998678
Prostate cancer; Preclinical model; Spheroids; Drug testing; SPOP
11.  Evaluation of ERG and SPINK1 by Immunohistochemical Staining and Clinicopathological Outcomes in a Multi-Institutional Radical Prostatectomy Cohort of 1067 Patients 
PLoS ONE  2015;10(7):e0132343.
Distinguishing between patients with early stage, screen detected prostate cancer who must be treated from those that can be safely watched has become a major issue in prostate cancer care. Identification of molecular subtypes of prostate cancer has opened the opportunity for testing whether biomarkers that characterize these subtypes can be used as biomarkers of prognosis. Two established molecular subtypes are identified by high expression of the ERG oncoprotein, due to structural DNA alterations that encode for fusion transcripts in approximately ½ of prostate cancers, and over-expression of SPINK1, which is purportedly found only in ERG-negative tumors. We used a multi-institutional prostate cancer tissue microarray constructed from radical prostatectomy samples with associated detailed clinical data and with rigorous selection of recurrent and non-recurrent cases to test the prognostic value of immunohistochemistry staining results for the ERG and SPINK1 proteins. In univariate analysis, ERG positive cases (419/1067; 39%) were associated with lower patient age, pre-operative serum PSA levels, lower Gleason scores (≤3+4=7) and improved recurrence free survival (RFS). On multivariate analysis, ERG status was not correlated with RFS, disease specific survival (DSS) or overall survival (OS). High-level SPINK1 protein expression (33/1067 cases; 3%) was associated with improved RFS on univariate and multivariate Cox regression analysis. Over-expression of either protein was not associated with clinical outcome. While expression of ERG and SPINK1 proteins was inversely correlated, it was not mutually exclusive since 3 (0.28%) cases showed high expression of both. While ERG and SPINK1 appear to identify discrete molecular subtypes of prostate cancer, only high expression of SPINK1 was associated with improved clinical outcome. However, by themselves, neither ERG nor SPINK1 appear to be useful biomarkers for prognostication of early stage prostate cancer.
doi:10.1371/journal.pone.0132343
PMCID: PMC4501723  PMID: 26172920
12.  Podoplanin increases migration and angiogenesis in malignant glioma 
Expression of podoplanin in glial brain tumors is grade dependent. While serving as a marker for tumor progression and modulating invasion in various neoplasms, little is known about podoplanin function in gliomas. Therefore we stably transfected two human glioma cell lines (U373MG and U87MG) with expression plasmids encoding podoplanin. The efficacy of transfection was confirmed by FACS analysis, PCR and immunocytochemistry. Cells were then sorted for highly podoplanin expressing cells (U373Phigh/U87Phigh). Transfection did not influence the production of pro-angiogenic factors including VEGF, VEGF-C and D. Also, expression of VEGF receptors (VEGFR) remained unchanged except for U87Phigh, where a VEGFR3 expression was induced. U373Phigh showed significantly reduced proliferation as compared to mock transfected group. By contrast, podoplanin significantly increased migration and invasion into collagen matrix. Furthermore, conditioned media from Phigh glioma cells strongly induced tube formation on matrigel. In conclusion, podoplanin increased migration of tumor cells and enhanced tube formation activity in endothelial cells independent from VEGF. Thus, podoplanin expression may be an important step in tumor progression.
PMCID: PMC4555782  PMID: 26339454
Podoplanin; glioma; angiogenesis
13.  Cellular Adhesion Promotes Prostate Cancer Cells Escape from Dormancy 
PLoS ONE  2015;10(6):e0130565.
Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of in vitro models. Here, we characterized in vitro PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant in vitro model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.
doi:10.1371/journal.pone.0130565
PMCID: PMC4475050  PMID: 26090669
14.  Testosterone Regulates Tight Junction Proteins and Influences Prostatic Autoimmune Responses 
Hormones & cancer  2011;2(3):145-156.
Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.
doi:10.1007/s12672-010-0063-1
PMCID: PMC4467375  PMID: 21761342
testosterone supplementation; inflammation; tight junction; claudin
15.  Neurodegeneration-associated instability of ribosomal DNA 
Biochimica et biophysica acta  2014;1842(6):860-868.
Homologous recombination (HR)-mediated instability of the repetitively organized ribosomal DNA (rDNA) has been proposed as a mediator of cell senescence in yeast triggering the DNA damage response. High individual variability in the content of human rDNA suggests that this genomic region remained relatively unstable throughout evolution. Therefore, quantitative real time PCR was used to determine the genomic content of rDNA in post mortem samples of parietal cortex from 14 young- and 9 elderly individuals with no diagnosis of a chronic neurodegenerative/neurological disease. In addition, rDNA content in that brain region was compared between 10 age-matched control individuals and 10 patients with dementia with Lewy bodies (DLB) which involves neurodegeneration of the cerebral cortex. Probing rRNA-coding regions of rDNA revealed no effects of aging on the rDNA content. Elevated rDNA content was observed in DLB. Conversely, in the DLB pathology-free cerebellum, lower genomic content of rDNA was present in the DLB group. In the parietal cortex, such a DLB-associated instability of rDNA was not accompanied by any major changes of CpG methylation of the rDNA promoter. As increased cerebro-cortical rDNA content was previously reported in Alzheimer's diseases, neurodegeneration appears to be associated with instability of rDNA. The hypothetical origins and consequences of this phenomenon are discussed including possibilities that the DNA damage-induced recombination destabilizes rDNA and that differential content of rDNA affects heterochromatin formation, gene expression and/or DNA damage response.
doi:10.1016/j.bbadis.2013.12.012
PMCID: PMC3985612  PMID: 24389328
Nucleolus; Genomic Instability; Dementia with Lewy Bodies; Neurodegeneration; Aging
16.  ABCC9 gene polymorphism is associated with hippocampal sclerosis of aging pathology 
Acta neuropathologica  2014;127(6):825-843.
Hippocampal sclerosis of aging (HS-Aging) is a high-morbidity brain disease in the elderly but risk factors are largely unknown. We report the first genome-wide association study (GWAS) with HS-Aging pathology as an endophenotype. In collaboration with the Alzheimer’s Disease Genetics Consortium, data were analyzed from large autopsy cohorts: (#1) National Alzheimer’s Coordinating Center (NACC); (#2) Rush University Religious Orders Study and Memory and Aging Project; (#3) Group Health Research Institute Adult Changes in Thought study; (#4) University of California at Irvine 90+ Study; and (#5) University of Kentucky Alzheimer’s Disease Center. Altogether, 363 HS-Aging cases and 2,303 controls, all pathologically confirmed, provided statistical power to test for risk alleles with large effect size. A two-tier study design included GWAS from cohorts #1–3 (Stage I) to identify promising SNP candidates, followed by focused evaluation of particular SNPs in cohorts #4–5 (Stage II). Polymorphism in the ATP-binding cassette, sub-family C member 9 (ABCC9) gene, also known as sulfonylurea receptor 2, was associated with HS-Aging pathology. In the meta-analyzed Stage I GWAS, ABCC9 polymorphisms yielded the lowest p values, and factoring in the Stage II results, the meta-analyzed risk SNP (rs704178:G) attained genome-wide statistical significance (p = 1.4 × 10−9), with odds ratio (OR) of 2.13 (recessive mode of inheritance). For SNPs previously linked to hippocampal sclerosis, meta-analyses of Stage I results show OR = 1.16 for rs5848 (GRN) and OR = 1.22 rs1990622 (TMEM106B), with the risk alleles as previously described. Sulfonylureas, a widely prescribed drug class used to treat diabetes, also modify human ABCC9 protein function. A subsample of patients from the NACC database (n = 624) were identified who were older than age 85 at death with known drug history. Controlling for important confounders such as diabetes itself, exposure to a sulfonylurea drug was associated with risk for HS-Aging pathology (p = 0.03). Thus, we describe a novel and targetable dementia risk factor.
doi:10.1007/s00401-014-1282-2
PMCID: PMC4113197  PMID: 24770881
Oldest old; Neuropathology; KATP; CTAGE5; ADGC; Potassium channel
17.  Disease-related microglia heterogeneity in the hippocampus of Alzheimer’s disease, dementia with Lewy bodies, and hippocampal sclerosis of aging 
Introduction
Neuropathological, genetic, and biochemical studies have provided support for the hypothesis that microglia participate in Alzheimer’s disease (AD) pathogenesis. Despite the extensive characterization of AD microglia, there are still many unanswered questions, and little is known about microglial morphology in other common forms of age-related dementia: particularly, dementia with Lewy bodies (DLB) and hippocampal sclerosis of aging (HS-Aging). In addition, no prior studies have attempted to compare and contrast the microglia morphology in the hippocampus of various neurodegenerative conditions.
Results
Here we studied cases with pathologically-confirmed AD (n = 7), HS-Aging (n = 7), AD + HS-aging (n = 4), DLB (n = 12), and normal (cognitively intact) controls (NC) (n = 9) from the University of Kentucky Alzheimer’s Disease Center autopsy cohort. We defined five microglia morphological phenotypes in the autopsy samples: ramified, hypertrophic, dystrophic, rod-shaped, and amoeboid. The Aperio ScanScope digital neuropathological tool was used along with two well-known microglial markers: IBA1 (a marker for both resting and activated microglia) and CD68 (a lysosomal marker in macrophages/microglia associated with phagocytic cells). Hippocampal staining analyses included studies of subregions within the hippocampal formation and nearby white matter. Using these tools and methods, we describe variation in microglial characteristics that show some degree of disease specificity, including, (1) increased microglia density and number in HS-aging and AD + HS-aging; (2) low microglia density in DLB; (3) increased number of dystrophic microglia in HS-aging; and (4) increased proportion of dystrophic to all microglia in DLB.
Conclusions
We conclude that variations in morphologies among microglial cells, and cells of macrophage lineage, can help guide future work connecting neuroinflammatory mechanisms with specific neurodegenerative disease subtypes.
doi:10.1186/s40478-015-0209-z
PMCID: PMC4489160  PMID: 26001591
Aging; Microglia activation; Mixed dementia; Neurodegeneration; Neuroinflammation; Neuropathology
18.  The Androgen-Regulated Protease TMPRSS2 Activates aProteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis 
Cancer discovery  2014;4(11):1310-1325.
TMPRSS2 is an androgen-regulated cell surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastasis. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-Met receptor tyrosine kinase signaling, and initiated a pro-invasive EMT phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment.
doi:10.1158/2159-8290.CD-13-1010
PMCID: PMC4409786  PMID: 25122198
prostate cancer; protease; invasion; metastasis; androgen receptor
19.  Development and Validation of a Scalable Next-Generation Sequencing System for Assessing Relevant Somatic Variants in Solid Tumors12 
Neoplasia (New York, N.Y.)  2015;17(4):385-399.
Next-generation sequencing (NGS) has enabled genome-wide personalized oncology efforts at centers and companies with the specialty expertise and infrastructure required to identify and prioritize actionable variants. Such approaches are not scalable, preventing widespread adoption. Likewise, most targeted NGS approaches fail to assess key relevant genomic alteration classes. To address these challenges, we predefined the catalog of relevant solid tumor somatic genome variants (gain-of-function or loss-of-function mutations, high-level copy number alterations, and gene fusions) through comprehensive bioinformatics analysis of >700,000 samples. To detect these variants, we developed the Oncomine Comprehensive Panel (OCP), an integrative NGS-based assay [compatible with < 20 ng of DNA/RNA from formalin-fixed paraffin-embedded (FFPE) tissues], coupled with an informatics pipeline to specifically identify relevant predefined variants and created a knowledge base of related potential treatments, current practice guidelines, and open clinical trials. We validated OCP using molecular standards and more than 300 FFPE tumor samples, achieving >95% accuracy for KRAS, epidermal growth factor receptor, and BRAF mutation detection as well as for ALK and TMPRSS2:ERG gene fusions. Associating positive variants with potential targeted treatments demonstrated that 6% to 42% of profiled samples (depending on cancer type) harbored alterations beyond routine molecular testing that were associated with approved or guideline-referenced therapies. As a translational research tool, OCP identified adaptive CTNNB1 amplifications/mutations in treated prostate cancers. Through predefining somatic variants in solid tumors and compiling associated potential treatment strategies, OCP represents a simplified, broadly applicable targeted NGS system with the potential to advance precision oncology efforts.
doi:10.1016/j.neo.2015.03.004
PMCID: PMC4415141  PMID: 25925381
AOHC, AcroMetrix Oncology Hotspot Control; CNAs, copy number alterations; FFPE, formalin-fixed paraffin-embedded; GoF, gain-of-function; indels, insertions/deletions; LoF, loss-of-function; LU, lung cohort; MCR, minimal common region; MO, molecular cohort; NCCN, National Comprehensive Cancer Network; NGS, next-generation sequencing; OCP, Oncomine Comprehensive Panel; PGM, Personal Genome Machine; PR, prostate cohort; QMRS, Quantitative Multiplex Reference Standard; SCC, small cell carcinoma; TCGA, The Cancer Genome Atlas
20.  Is synaptic loss a unique hallmark of Alzheimer's disease? 
Biochemical pharmacology  2014;88(4):517-528.
Synapses may represent a key nidus for dementia including Alzheimer's disease (AD) pathogenesis. Here we review published studies and present new ideas related to the question of the specificity of synapse loss in AD. Currently, AD is defined by the regional presence of neuritic plaques and neurofibrillary tangles in the brain. The severity of involvement by those pathological hallmarks tends to correlate both with antemortem cognitive status, and also with synapse loss in multiple brain areas. Recent studies from large autopsy series have led to a new standard of excellence with regard to clinical–pathological correlation and to improved comprehension of the numerous brain diseases of the elderly. These studies have provided evidence that it is the rule rather than the exception for brains of aged individuals to demonstrate pathologies (often multiple) other than AD plaques and tangles. For many of these comorbid pathologies, the extent of synapse loss is imperfectly understood but could be substantial. These findings indicate that synapse loss is probably not a hallmark specific to AD but rather a change common to many diseases associated with dementia.
doi:10.1016/j.bcp.2013.12.028
PMCID: PMC4230706  PMID: 24412275
TDP-43; Neuropathology; Cerebrovascular; Hippocampal sclerosis; HS-Aging
21.  Impact of Cultivation and Subsequent Burial on Cydia pomonella (Lepidoptera: Tortricidae) and Conotrachelus nenuphar (Coleoptera: Curculionidae) 
Journal of Economic Entomology  2015;108(3):1215-1220.
We assessed the efficacy of cultivation as a potential management strategy for codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae), and plum curculio, Conotrachelus nenuphar Herbst (Coleoptera: Curculionidae) in apple orchards. Cocooned codling moth pupae and thinning apples infested with plum curculio larvae were cultivated over in the field. Emergence, percent burial, damage to buried fruit, and depth of burial was recorded. In the laboratory, both insects were buried at variable depths in sand and potting soil and emergence was measured. A greater proportion of plum curculio larvae buried in infested fruit under laboratory conditions survived to adulthood compared with unburied infested fruit, down to 15 cm. No codling moth adults emerged from under 1 cm or more of sand. Buried codling moth larvae experienced drastically reduced survival to adulthood compared with unburied larvae. These results indicate that strip cultivation may negatively impact codling moth diapausing larvae and pupae on the ground, but not likely to negatively impact plum curculio in infested dropped apples.
doi:10.1093/jee/tov071
PMCID: PMC4477519  PMID: 26470248
strip cultivation; codling moth; Cydia pomonella; plum curculio; Conotrachelus nenuphar
22.  Treatment of advanced gastrointestinal tumors with genetically modified autologous mesenchymal stromal cells (TREAT-ME1): study protocol of a phase I/II clinical trial 
BMC Cancer  2015;15:237.
Background
Adenocarcinoma originating from the digestive system is a major contributor to cancer-related deaths worldwide. Tumor recurrence, advanced local growth and metastasis are key factors that frequently prevent these tumors from curative surgical treatment. Preclinical research has demonstrated that the dependency of these tumors on supporting mesenchymal stroma results in susceptibility to cell-based therapies targeting this stroma.
Methods/Design
TREAT-ME1 is a prospective, uncontrolled, single-arm phase I/II study assessing the safety and efficacy of genetically modified autologous mesenchymal stromal cells (MSC) as delivery vehicles for a cell-based gene therapy for advanced, recurrent or metastatic gastrointestinal or hepatopancreatobiliary adenocarcinoma. Autologous bone marrow will be drawn from each eligible patient after consent for bone marrow donation has been obtained (under a separate EC-approved protocol). In the following ~10 weeks the investigational medicinal product (IMP) is developed for each patient. To this end, the patient’s MSCs are stably transfected with a gamma-retroviral, replication-incompetent and self-inactivating (SIN) vector system containing a therapeutic promoter - gene construct that allows for tumor-specific expression of the therapeutic gene. After release of the IMP the patients are enrolled after given informed consent for participation in the TREAT-ME 1 trial. In the phase I part of the study, the safety of the IMP is tested in six patients by three treatment cycles consisting of re-transfusion of MSCs at different concentrations followed by administration of the prodrug Ganciclovir. In the phase II part of the study, sixteen patients will be enrolled receiving IMP treatment. A subgroup of patients that qualifies for surgery will be treated preoperatively with the IMP to verify homing of the MSCs to tumors as to be confirmed in the surgical specimen.
Discussion
The TREAT-ME1 clinical study involves a highly innovative therapeutic strategy combining cell and gene therapy and is conducted at a high level of pharmaceutical quality ensuring patient safety. This patient-tailored approach represents the first clinical study worldwide utilizing genetically engineered MSCs in humans.
Trial registration
EU Clinical Trials Register/European Union Drug Regulating Authorities Clinical Trials Database number: 2012-003741-15
doi:10.1186/s12885-015-1241-x
PMCID: PMC4393860  PMID: 25879229
Genetically engineered mesenchymal stromal cells; MSC; Gene therapy; Cell therapy; Suicide gene therapy; Clinical trial; HSV-Tk; Ganciclovir
23.  Assessing the Discriminant Ability, Reliability, and Comparability of Multiple Short Forms of the Boston Naming Test in an Alzheimer’s Disease Center Cohort 
Background
The Boston Naming Test (BNT) is a commonly used neuropsychological test of confrontation naming that aids in determining the presence and severity of dysnomia. Many short versions of the original 60-item test have been developed and are routinely administered in clinical/research settings. Because of the common need to translate similar measures within and across studies, it is important to evaluate the operating characteristics and agreement of different BNT versions.
Methods
We analyzed longitudinal data of research volunteers (n = 681) from the University of Kentucky Alzheimer’s Disease Center longitudinal cohort.
Conclusions
With the notable exception of the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) 15-item BNT, short forms were internally consistent and highly correlated with the full version; these measures varied by diagnosis and generally improved from normal to mild cognitive impairment (MCI) to dementia. All short forms retained the ability to discriminate between normal subjects and those with dementia. The ability to discriminate between normal and MCI subjects was less strong for the short forms than the full BNT, but they exhibited similar patterns. These results have important implications for researchers designing longitudinal studies, who must consider that the statistical properties of even closely related test forms may be quite different.
doi:10.1159/000370108
PMCID: PMC4374652  PMID: 25613081
Alzheimer’s disease; Cognitive impairment; Cohort studies; Clinical diagnosis; Clinical neuropsychology; Dementia and neuropsychology; Design, analysis, interpretation of data; Longitudinal assessment; Mild cognitive impairment and dementia; Neuropsychiatric assessment
24.  Rapid Induction of Androgen Receptor Splice Variants by Androgen Deprivation in Prostate Cancer 
Purpose
Mechanisms mediating androgen receptor (AR) reactivation in prostate cancer (PCa) that progresses after castration (castration-resistant prostate cancer, CRPC) and subsequent treatment with abiraterone (CYP17A1 inhibitor that further suppresses androgen synthesis) remain unclear.
Experimental Design
PCa xenografts were examined to identify mechanism of progression after castration and abiraterone.
Results
AR reactivation in abiraterone-resistant VCaP xenografts was not associated with restoration of intratumoral androgens or alterations in AR co-regulators. In contrast, mRNA encoding full length AR (AR-FL) and a constitutively active splice variant (AR-V7) were increased compared to xenografts prior to castration, with an increase in AR-V7 relative to AR-FL. This shift towards AR-V7 was due to a feedback mechanism whereby the androgen-liganded AR stimulates expression of proteins that suppress generation of AR-V7 relative to AR-FL transcripts. However, despite the increases in AR-V7 mRNA, it remained a minor transcript (<1%) relative to AR-FL in resistant VCaP xenografts and CRPC clinical samples. AR-V7 protein expression was similarly low relative to AR-FL in castration-resistant VCaP xenografts and androgen-deprived VCaP cells, but the weak basal AR activity in these latter cells was further repressed by AR-V7 siRNA.
Conclusions
AR-V7 at these low levels is not adequate to restore AR activity, but its rapid induction after androgen deprivation allows tumors to retain basal AR activity that may be needed for survival until more potent mechanisms emerge to activate AR. Agents targeting AR splice variants may be most effective when used very early in conjunction with therapies targeting the AR ligand binding domain.
doi:10.1158/1078-0432.CCR-13-1863
PMCID: PMC4022291  PMID: 24449822

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