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1.  Hippocampal Sclerosis in Dementia, Epilepsy, and Ischemic Injury: Differential Vulnerability of Hippocampal Subfields 
Severe neuronal loss in the hippocampus, that is, hippocampal sclerosis (HS), can be seen in 3 main clinical contexts: dementia (particularly frontotemporal lobar degeneration [FTLD]), temporal lobe epilepsy (TLE), and hippocampal ischemic injury (H–I). It has been suggested that shared pathogenetic mechanisms may underlie selective vulnerability of the hippocampal subfields such as the CA1 in these conditions. We determined the extent of neuronal loss in cases of HS-FTLD (n = 14), HS-TLE (n = 35), and H–I (n = 20). Immunohistochemistry for zinc transporter 3 was used to help define the CA3/CA2 border in the routinely processed human autopsy tissue samples. The subiculum was involved in 57% of HS-FTLD, 10% of H–I, and 0% of HS-TLE cases (p < 0.0001). The CA regions other than CA1 were involved in 57% of HS-TLE, 30% of H–I, and 0% of HS-FTLD cases (p= 0.0003). The distal third of CA1 was involved in 79% of HS-FTLD, 35% of H–I, and 37% of HS-TLE cases (p = 0.02). The distal third of CA1 was the only area involved in 29% of HS-FTLD, 3% of HS-TLE, and 0% of H–I cases (p = 0.019). The proximal-middle CA1 was the only area affected in 50% of H–I, 29% of HS-TLE, and 0% of HS-FTLD cases (p = 0.004). These findings support heterogeneity in the pathogenesis of HS.
doi:10.1097/OPX.0000000000000170
PMCID: PMC4076969  PMID: 24423638
Brain ischemia; Frontotemporal dementia; Frontotemporal lobar degeneration; Hippocampal sclerosis; Mesial temporal sclerosis; Vascular dementia; ZNT3
2.  Involvement of estrogen receptor b5 in the progression of glioma 
Brain research  2013;1503:97-107.
Emerging evidence suggests a decline of ERβ expression in various peripheral cancers. ERβ has been proposed as a cancer brake that inhibits tumor proliferation. In the current study, we have identified ERβ5 as the predominant isoform of ERβ in human glioma and its expression was significantly increased in human glioma as compared with non-neoplastic brain tissue. Hypoxia and activation of hypoxia inducible factor (HIF) increased ERβ transcription in U87 cells, suggesting elevated ERβ expression in glioma might be induced by the hypoxic stress in the tumor. Over-expression of either ERβ1 or ERβ5 increased PTEN expression and inhibited activation of the PI3K/AKT/mTOR pathway. In addition, ERβ5 inhibited the MAPK/ERK pathway. In U87 cells, ERβ1 and ERβ5 inhibit cell proliferation and reduced cells in the S+G2/M phase. Our findings suggest hypoxia induced ERβ5 expression in glioma as a self-protective mechanism against tumor proliferation and that ERβ5 might serve as a therapeutic target for the treatment of glioma.
doi:10.1016/j.brainres.2013.02.004
PMCID: PMC3646566  PMID: 23399685
Estrogen receptor β; Glioblastoma multiforme; Hypoxia inducible factor; PTEN
3.  Length of normal alleles of C9ORF72 GGGGCC repeat do not influence disease phenotype 
Neurobiology of aging  2012;33(12):2950.e5-2950.e7.
Expansions of the non-coding GGGGCC hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene were recently identified as the long sought-after cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) on chromosome 9p. In this study we aimed to determine whether the length of the normal - unexpanded - allele of the GGGGCC repeat in C9ORF72 plays a role in the presentation of disease or affects age at onset in C9ORF72 mutation carriers. We also studied whether the GGGGCC repeat length confers risk or affects age at onset in FTD and ALS patients without C9ORF72 repeat expansions. C9ORF72 genotyping was performed in 580 FTD, 995 ALS and 160 FTD-ALS patients and 1444 controls, leading to the identification of 211 patients with pathogenic C9ORF72 repeat expansions and an accurate quantification of the length of the normal alleles in all patients and controls. No meaningful association between the repeat length of the normal alleles of the GGGGCC repeat in C9ORF72 and disease phenotype or age at onset was observed in C9ORF72 mutation carriers or non-mutation carriers.
doi:10.1016/j.neurobiolaging.2012.07.005
PMCID: PMC3617405  PMID: 22840558
Amyotrophic lateral sclerosis; Frontotemporal Dementia; C9ORF72; Repeat-expansion disease; Association study
4.  Opposing effect of EGFRwt on EGFRvIII-mediated NF-κB activation with RIP1 as a cell death switch 
Cell reports  2013;4(4):764-775.
Summary
RIP1 is a central mediator of cell death in cell stress, but can also mediate cell survival by activating NF-κB. Here, we show that RIP1 is a switch in EGFR signaling. EGFRvIII is an oncogenic mutant that does not bind ligand and is co-expressed with EGFRwt in glioblastoma (GBM). EGFRvIII recruits ubiquitin ligases to RIP1 resulting in K63-linked ubiquitination of RIP1. RIP1 binds to TAK1 and NEMO forming a EGFRvIII-RIP1 signalosome that activates NF-κB. RIP1 is essential for EGFRvIII-mediated oncogenicity and correlates with NF-κB activation in GBM. Surprisingly, activation of EGFRwt with EGF results in a novel negative regulation of EGFRvIII with dissociation of the EGFRvIII-RIP1 signalosome, loss of RIP1 ubiquitination, NF-κB activation, and association of RIP1 with FADD and Caspase-8. If EGFRwt is overexpressed with EGFRvIII, adding EGF leads to a RIP1 kinase dependent cell death. The EGFRwt-EGFRvIII-RIP1 interplay may regulate oncogenicity and vulnerability to targeted treatment in GBM.
doi:10.1016/j.celrep.2013.07.025
PMCID: PMC3780989  PMID: 23972990
EGFRvIII; glioblastoma; EGFR wild type; NF-kappaB; RIP1; NEMO; TAK1; antagonism
5.  Regional changes of cortical mean diffusivities with aging after correction of partial volume effects 
NeuroImage  2012;62(3):1705-1716.
Accurately measuring the cortical mean diffusivity (MD) derived from diffusion tensor imaging (DTI) at the comprehensive lobe, gyral and voxel level of young, elderly healthy brains and those with Alzheimer's disease (AD) may provide insights on heterogeneous cortical microstructural changes caused by aging and AD. Due to partial volume effects (PVE), the measurement of cortical MD is overestimated with contamination of cerebrospinal fluid (CSF). The bias is especially severe for aging and AD brains because of significant cortical thinning of these brains. In this study, we aimed to quantitatively characterize the unbiased regional cortical MD changes due to aging and AD and delineate the effects of cortical thinning of elderly healthy and AD groups on MD measurements. DTI and T1-weighted images of 14 young, 15 elderly healthy subjects and 17 AD patients were acquired. With the parcellated cortical gyri and lobes from T1 weighted image transformed to DTI, regional cortical MD of all subjects before and after PVE correction were measured. CSF contamination model was used to correct bias of MD caused by PVE. Compared to cortical MD of young group, significant increases of corrected MD for elderly healthy and AD groups were found only in frontal and limbic regions, respectively, while there were significant increases of uncorrected MD all over the cortex. Uncorrected MD are significantly higher in limbic and temporal gyri in AD group, compared to those in elderly healthy group but higher MD only remained in limbic gyri after PVE correction. Cortical thickness was also measured for all groups. The correlation slopes between cortical MD and thickness for elderly healthy and AD groups were significantly decreased after PVE correction compared to before correction while no significant change of correlation slope was detected for young group. It suggests that the cortical thinning in elderly healthy and AD groups is a significant contributor to the bias of uncorrected cortical MD measurement. The established comprehensive unbiased cortical MD profiles of young, elderly healthy subjects and AD patients at the lobe, gyral and voxel level may serve as clinical references for cortical microstructure.
doi:10.1016/j.neuroimage.2012.05.082
PMCID: PMC3574164  PMID: 22683383
DTI; Cortex; Mean diffusivity; Aging; Alzheimer's disease; Unbiased; Partial volume effects
6.  TREM2 in neurodegeneration: evidence for association of the p.R47H variant with frontotemporal dementia and Parkinson’s disease 
Background
A rare variant in the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) gene has been reported to be a genetic risk factor for Alzheimer’s disease by two independent groups (Odds ratio between 2.9-4.5). Given the key role of TREM2 in the effective phagocytosis of apoptotic neuronal cells by microglia, we hypothesized that dysfunction of TREM2 may play a more generalized role in neurodegeneration. With this in mind we set out to assess the genetic association of the Alzheimer’s disease-related risk variant in TREM2 (rs75932628, p.R47H) with other related neurodegenerative disorders.
Results
The study included 609 patients with frontotemporal dementia, 765 with amyotrophic lateral sclerosis, 1493 with Parkinson’s disease, 772 with progressive supranuclear palsy, 448 with ischemic stroke and 1957 controls subjects free of neurodegenerative disease. A significant association was observed for the TREM2 p.R47H substitution in susceptibility to frontotemporal dementia (OR = 5.06; p-value = 0.001) and Parkinson’s disease (OR = 2.67; p-value = 0.026), while no evidence of association with risk of amyotrophic lateral sclerosis, progressive supranuclear palsy or ischemic stroke was observed.
Conclusions
Our results suggest that the TREM2 p.R47H substitution is a risk factor for frontotemporal dementia and Parkinson’s disease in addition to Alzheimer’s disease. These findings suggest a more general role for TREM2 dysfunction in neurodegeneration, which could be related to its role in the immune response.
doi:10.1186/1750-1326-8-19
PMCID: PMC3691612  PMID: 23800361
TREM2; Frontotemporal dementia; Parkinson disease; Genetic association
7.  Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse, human glioblastomas in the mouse brain in vivo 
Cell Metabolism  2012;15(6):827-837.
SUMMARY
Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused 13C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.
doi:10.1016/j.cmet.2012.05.001
PMCID: PMC3372870  PMID: 22682223
8.  Glucose Metabolism via the Pentose Phosphate Pathway, Glycolysis and Krebs Cycle in an Orthotopic Mouse Model of Human Brain Tumors 
NMR in biomedicine  2012;25(10):1177-1186.
It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using an orthotopic mouse model of primary human glioblastoma (GBM) and a brain metastatic renal tumor of clear cell renal cell carcinoma (CCRCC) histology, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-13C2]glucose. The [3-13C]lactate/[2,3-13C2]lactate ratio was similar for both the GBM and renal tumor and their respective surrounding brains (GBM: 0.197 ± 0.011 and 0.195 ± 0.033 (p=1); CCRCC: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than PPP flux in these tumors, and that PPP flux into the lactate pool was similar in both tissues. Remarkably, 13C-13C coupling was observed in molecules derived from Krebs cycle intermediates in both tumors, denoting glucose oxidation. In the renal tumor, in contrast to GBM and surrounding brain, 13C multiplets of GABA differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. Additionally, the orthotopic renal tumor, the patient’s primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a CCRCC tissue microarray suggesting that GABA synthesis is cell-autonomous in at least a subset of renal tumors. Taken together, these data demonstrate that 13C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.
doi:10.1002/nbm.2787
PMCID: PMC3670098  PMID: 22383401
Intermediary metabolism; Mouse model; 13C NMR; brain; glioblastoma; renal cell carcinoma
9.  Metabolism of [U-13C]glucose in Human Brain Tumors In Vivo 
NMR in biomedicine  2012;25(11):1234-1244.
Glioblastomas (GBMs) and brain metastases demonstrate avid uptake of 18fluoro-2-deoxyglucose (FDG) by positron emission tomography (PET) and display perturbations of intracellular metabolite pools by 1H magnetic resonance spectroscopy (MRS). These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. FDG-positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation compared to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain malignancies to oxidize glucose in the tricarboxylic acid cycle is unknown. Here we studied the metabolism of human brain tumors in situ. [U-13C]glucose was infused during surgical resection, and tumor samples were subsequently subjected to 13C NMR spectroscopy. Analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the TCA cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl-CoA pool was derived from blood-borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of 13C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse malignancies growing in their native microenvironment.
doi:10.1002/nbm.2794
PMCID: PMC3406255  PMID: 22419606
Cancer; glioblastoma; metabolism; Warburg effect; glucose; glutamine; NMR
10.  Ataxin-2 repeat-length variation and neurodegeneration 
Human Molecular Genetics  2011;20(16):3207-3212.
Expanded glutamine repeats of the ataxin-2 (ATXN2) protein cause spinocerebellar ataxia type 2 (SCA2), a rare neurodegenerative disorder. More recent studies have suggested that expanded ATXN2 repeats are a genetic risk factor for amyotrophic lateral sclerosis (ALS) via an RNA-dependent interaction with TDP-43. Given the phenotypic diversity observed in SCA2 patients, we set out to determine the polymorphic nature of the ATXN2 repeat length across a spectrum of neurodegenerative disorders. In this study, we genotyped the ATXN2 repeat in 3919 neurodegenerative disease patients and 4877 healthy controls and performed logistic regression analysis to determine the association of repeat length with the risk of disease. We confirmed the presence of a significantly higher number of expanded ATXN2 repeat carriers in ALS patients compared with healthy controls (OR = 5.57; P= 0.001; repeat length >30 units). Furthermore, we observed significant association of expanded ATXN2 repeats with the development of progressive supranuclear palsy (OR = 5.83; P= 0.004; repeat length >30 units). Although expanded repeat carriers were also identified in frontotemporal lobar degeneration, Alzheimer's and Parkinson's disease patients, these were not significantly more frequent than in controls. Of note, our study identified a number of healthy control individuals who harbor expanded repeat alleles (31–33 units), which suggests caution should be taken when attributing specific disease phenotypes to these repeat lengths. In conclusion, our findings confirm the role of ATXN2 as an important risk factor for ALS and support the hypothesis that expanded ATXN2 repeats may predispose to other neurodegenerative diseases, including progressive supranuclear palsy.
doi:10.1093/hmg/ddr227
PMCID: PMC3140823  PMID: 21610160
11.  Genetic and Clinical Features of Progranulin-Associated Frontotemporal Lobar Degeneration 
Archives of neurology  2011;68(4):488-497.
Objective
To assess the relative frequency of unique mutations and their associated characteristics in 97 individuals with mutations in progranulin (GRN), an important cause of frontotemporal lobar degeneration (FTLD).
Participants and Design
A 46-site International Frontotemporal Lobar Degeneration Collaboration was formed to collect cases of FTLD with TAR DNA-binding protein of 43-kDa (TDP-43)–positive inclusions (FTLD-TDP). We identified 97 individuals with FTLD-TDP with pathogenic GRN mutations (GRN+ FTLD-TDP), assessed their genetic and clinical characteristics, and compared them with 453 patients with FTLD-TDP in which GRN mutations were excluded (GRN− FTLD-TDP). No patients were known to be related. Neuropathologic characteristics were confirmed as FTLD-TDP in 79 of the 97 GRN+ FTLDTDP cases and all of the GRN− FTLD-TDP cases.
Results
Age at onset of FTLD was younger in patients with GRN+ FTLD-TDP vs GRN− FTLD-TDP (median, 58.0 vs 61.0 years; P<.001), as was age at death (median, 65.5 vs 69.0 years; P<.001). Concomitant motor neuron disease was much less common in GRN+ FTLDTDP vs GRN− FTLD-TDP (5.4% vs 26.3%; P<.001). Fifty different GRN mutations were observed, including 2 novel mutations: c.139delG (p.D47TfsX7) and c.378C>A (p.C126X). The 2 most common GRN mutations were c.1477C>T (p.R493X, found in 18 patients, representing 18.6% of GRN cases) and c.26C>A (p.A9D, found in 6 patients, representing 6.2% of cases). Patients with the c.1477C>T mutation shared a haplotype on chromosome 17; clinically, they resembled patients with other GRN mutations. Patients with the c.26C>A mutation appeared to have a younger age at onset of FTLD and at death and more parkinsonian features than those with other GRN mutations.
Conclusion
GRN+ FTLD-TDP differs in key features from GRN− FTLD-TDP.
doi:10.1001/archneurol.2011.53
PMCID: PMC3160280  PMID: 21482928
12.  The Telomerase Antagonist Imetelstat Efficiently Targets Glioblastoma Tumor-Initiating Cells Leading to Decreased Proliferation and Tumor Growth 
Purpose
Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo.
Experimental design
Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat.
Results
Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC50 0.45μM). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg q3d) showed ∼70% inhibition of telomerase activity. Chronic systemic treatment on the same dose schedule produced a marked decrease in the rate of xenograft subcutaneous tumor growth.
Conclusion
This pre-clinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies.
doi:10.1158/1078-0432.CCR-09-2850
PMCID: PMC2883447  PMID: 20048334
neurospheres; telomeres; temozolomide; radiation; orthotopic
13.  Epidermal Growth Factor Receptor in Glioma: Signal Transduction, Neuropathology, Imaging, and Radioresistance1 
Neoplasia (New York, N.Y.)  2010;12(9):675-684.
Aberrant epidermal growth factor receptor (EGFR) signaling is common in cancer. Increased expression of wild type and mutant EGFR is a widespread feature of diverse types of cancer. EGFR signaling in cancer has been the focus of intense investigation for decades primarily for two reasons. First, aberrant EGFR signaling is likely to play an important role in the pathogenesis of cancer, and therefore, the mechanisms of EGFR-mediated oncogenic signaling are of interest. Second, the EGFR signaling system is an attractive target for therapeutic intervention. EGFR gene amplification and overexpression are a particularly striking feature of glioblastoma (GBM), observed in approximately 40% of tumors. GBM is the most common primary malignant tumor of the central nervous system in adults. In approximately 50% of tumors with EGFR amplification, a specific EGFR mutant (EGFRvIII, also known as EGFR type III, de2-7, ΔEGFR) can be detected. This mutant is highly oncogenic and is generated from a deletion of exons 2 to 7 of the EGFR gene, which results in an in-frame deletion of 267 amino acids from the extracellular domain of the receptor. EGFRvIII is unable to bind ligand, and it signals constitutively. Although EGFRvIII has the same signaling domain as the wild type receptor, it seems to generate a distinct set of downstream signals that may contribute to an increased tumorigenicity. In this review, we discuss recent progress in key aspects of EGFR signaling in GBM, focusing on neuropathology, signal transduction, imaging of the EGFR, and the role of the EGFR in mediating resistance to radiation therapy in GBM.
PMCID: PMC2933688  PMID: 20824044
14.  TDP-43 pathology in primary progressive aphasia and frontotemporal dementia with pathologic Alzheimer disease 
Acta neuropathologica  2010;120(1):43-54.
The clinical syndrome of primary progressive aphasia (PPA) can be associated with a variety of neuropathologic diagnoses at autopsy. Thirty percent of cases have Alzheimer disease (AD) pathology, most often in the usual distribution, which defies principles of brain–behavior organization, in that aphasia is not symptomatic of limbic disease. The present study investigated whether concomitant TDP-43 pathology could resolve the lack of clinicoanatomic concordance. In this paper, 16 cases of clinical PPA and 10 cases of primarily non-aphasic frontotemporal dementia (FTD), all with AD pathology, were investigated to determine whether their atypical clinical phenotypes reflected the presence of additional TDP-43 pathology. A comparison group consisted of 27 cases of pathologic AD with the typical amnestic clinical phenotype of probable AD. Concomitant TDP-43 pathology was discovered in only three of the FTD and PPA but in more than half of the typical amnestic clinical phenotypes. Hippocampal sclerosis (HS) was closely associated with TDP-43 pathology when all groups were combined for analysis. Therefore, the clinical phenotypes of PPA and FTD in cases with pathologic AD are only rarely associated with TDP-43 proteinopathy. Furthermore, medial temporal TDP-43 pathology is more tightly linked to HS than to clinical phenotype. These findings challenge the current notions about clinicopathologic correlation, especially about the role of multiple pathologies.
doi:10.1007/s00401-010-0681-2
PMCID: PMC2903745  PMID: 20361198
Primary progressive aphasia; Frontotemporal dementia; Alzheimer disease; FTLD-TDP; TDP-43 proteinopathy; Hippocampal sclerosis
15.  RIP1 activates PI3K-Akt via a dual mechanism involving NF-kappaB mediated inhibition of the mTOR-S6K-IRS1 negative feedback loop and downregulation of PTEN 
Cancer research  2009;69(10):4107-4111.
Summary
Therapeutic inhibition of mTOR in cancer is complicated by the existence of a negative feedback loop linking mTOR to the PI3K-Akt pathway. Thus, mTOR inhibition by Rapamycin or TSC1/2 results in increased PI3K-Akt activation. The death domain kinase receptor interacting protein 1 (RIP, RIP1), plays a key role in NF-κB activation and also activates the PI3K-Akt pathway through unknown mechanisms. RIP1 has recently been found to be overexpressed in glioblastoma (GBM), the most common adult primary malignant brain tumor, but not in Grade II-III glioma. Our data suggest that RIP1 activates PI3K-Akt using dual mechanisms by removing the two major brakes on PI3K-Akt activity. Firstly, increased expression of RIP1 activates PI3K-Akt by interrupting the mTOR negative feedback loop. However, unlike other signals which regulate mTOR activity without affecting its level, RIP1 negatively regulates mTOR transcription via a NF-κB dependent mechanism. The second mechanism used by RIP1 to activate PI3K-Akt is downregulation of cellular PTEN levels which appears to be independent of NF-κB activation. The clinical relevance of these findings is highlighted by the demonstration that RIP1 levels correlate with activation of Akt in GBM. Thus, our study shows that RIP1 regulates key components of the PTEN-PI3K-Akt-mTOR pathway and elucidates a novel negative regulation of mTOR signaling at the transcriptional level by the NF-κB pathway. Our data suggest that the RIP1-NF-κB status of tumors may influence response to treatments targeting the PTEN-PI3K-mTOR signaling axis.
doi:10.1158/0008-5472.CAN-09-0474
PMCID: PMC2683913  PMID: 19435890
PI3K-Akt; mammalian target of rapamycin; receptor interacting protein; NF-kappa B; negative feedback
16.  EGFRvIII and DNA Double-Strand Break Repair: A Molecular Mechanism for Radioresistance in Glioblastoma 
Cancer research  2009;69(10):4252-4259.
Glioblastoma multiforme (GBM) are lethal tumors that are highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, EGFRvIII, confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSBs), the most lethal lesion inflicted by IR. The EGFR inhibitor Gefitinib (Iressa) and the PI3K inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated-Akt-1 accelerates repair, implicating the PI3K-Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-PKcs. Enhanced radioresistance is abrogated by the DNA-PKcs-specific inhibitor NU7026 and EGFRvIII fails to confer radioresistance in DNA-PKcs-deficient cells. In vivo, orthotopic U87-EGFRvIII-derived tumors display faster rates of DSB repair following whole brain radiotherapy (WBRT) compared to U87-derived tumors. Consequently, EGFRVIII-expressing tumors are radioresistant and continue to grow following WBRT with little impact on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII-expression promotes DNA-PKcs activation and DSB repair, perhaps, as a consequence of hyperactivated PI3K-Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas.
doi:10.1158/0008-5472.CAN-08-4853
PMCID: PMC2694953  PMID: 19435898
glioblastoma multiforme (GBM); radioresistance; DNA double-strand break (DSB); DNA repair; epidermal growth factor receptor (EGFR); DNA-dependent protein kinase (DNA-PK)
17.  Concurrent Vestibular Schwannoma and Meningioma Mimicking a Single Cerebellopontine Angle Tumor 
Skull Base  2009;19(6):443-446.
ABSTRACT
Vestibular schwannomas account for ~;80% of cerebellopontine angle (CPA) tumors, with meningiomas being the second most common tumor of the CPA. The occurrence of both a schwannoma and a meningioma in the cerebellopontine angle is rare. After obtaining Institutional Review Board approval, we present a case of a concurrent vestibular schwannoma and meningioma in the CPA mimicking a single tumor.
doi:10.1055/s-0029-1220206
PMCID: PMC2793893  PMID: 20436847
Acoustic neuroma; vestibular schwannoma; meningioma; cerebellopontine angle
18.  The receptor interacting protein (RIP1) inhibits p53 induction through NF-κB activation and confers a worse prognosis in glioblastoma 
Cancer research  2009;69(7):2809-2816.
NF-κB activation may play an important role in the pathogenesis of cancer and also in resistance to treatment. Inactivation of the p53 tumor suppressor is a key component of the multi-step evolution of most cancers. Links between the NF-κB and p53 pathways are under intense investigation. In this study, we show that the receptor interacting protein (RIP, RIP1), a central component of the NF-κB signaling network, negatively regulates p53 tumor suppressor signaling. Loss of RIP1 from cells results in augmented induction of p53 in response to DNA damage, while increased RIP1 level leads to a complete shutdown of DNA-damage induced p53 induction by enhancing levels of cellular mdm2. The key signal generated by RIP1 to upregulate mdm2 and inhibit p53 is activation of NF-κB. The clinical implication of this finding is demonstrated in glioblastoma (GBM), the most common primary malignant brain tumor in adults. We show that RIP1 is commonly overexpressed in GBM but not in grade II-III glioma and increased expression of RIP1 confers a worse prognosis in GBM. Importantly, RIP1 levels correlate strongly with mdm2 levels in GBM. Our results demonstrate a key interaction between the NF-κB and p53 pathways that may have implications for the targeted treatment of GBM.
doi:10.1158/0008-5472.CAN-08-4079
PMCID: PMC2859885  PMID: 19339267
inflammation; cancer; p53; RIP1; NF-kappa B; prognosis; glioblastoma
19.  Molecular Characterization of Novel Progranulin (GRN) Mutations in Frontotemporal Dementia 
Human mutation  2008;29(4):512-521.
Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3′ splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6–2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency.
doi:10.1002/humu.20681
PMCID: PMC2756561  PMID: 18183624
Frontotemporal dementia; FTD; granulin; progranulin; GRN; PGRN
20.  TDP-43 A315T Mutation in Familial Motor Neuron Disease 
Annals of neurology  2008;63(4):535-538.
To identify novel causes of familial neurodegenerative diseases, we extended our previous studies of TAR DNA-binding protein 43 (TDP-43) proteinopathies to investigate TDP-43 as a candidate gene in familial cases of motor neuron disease. Sequencing of the TDP-43 gene led to the identification of a novel missense mutation, Ala-315-Thr, which segregates with all affected members of an autosomal dominant motor neuron disease family. The mutation was not found in 1,505 healthy control subjects. The discovery of a missense mutation in TDP-43 in a family with dominantly inherited motor neuron disease provides evidence of a direct link between altered TDP-43 function and neurodegeneration.
doi:10.1002/ana.21344
PMCID: PMC2747362  PMID: 18288693
21.  TDP-43 immunohistochemistry reveals extensive neuritic pathology in FTLD-U: a Midwest-Southwest Consortium for FTLD study 
TDP-43 is a major component of the inclusions in frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). We studied TDP-43-pathology in the hippocampus and frontal cortex of autopsy brains with FTLD-U (n=68), dementia lacking distinctive histopathology (n=4), other neurodegenerative diseases (n=23), and controls (n=12). A marked enhancement of TDP-43-positive dystrophic neurites (DN) was obtained by using a sensitive immunohistochemistry protocol. Two previously unrecognized patterns of pathology were observed: frequent long DN in the CA1 region and frequent dot-like DN in the neocortical layer II, which were seen in 39% and 15% of the FTLD-U cases, respectively. Four FTLD-U cases showed no TDP-43 pathology and were reclassified as FTLD-U, non-TDP-43 proteinopathy. Frequent long DN, but not dot-like DN, were significantly associated with progranulin mutations. Three of the DLDH cases were reclassified as FTLD-U. Of the cases with other neurodegenerative diseases, 43% showed TDP-43-pathology in the hippocampus but only 4% in the frontal cortex. No TDP-43-pathology was seen in controls. These results indicate that the sensitivity of the TDP-43 immunohistochemistry method affects both the quantity of the pathology and the types of pathology that can be detected. Involvement of both the hippocampus and frontal cortex may be a diagnostically important feature in FTLD-U.
doi:10.1097/NEN.0b013e31816a12a6
PMCID: PMC2635119  PMID: 18379440
Frontotemporal lobar degeneration with ubiquitinated inclusions; frontotemporal lobar degeneration with motor neuron disease; dementia lacking distinctive histopathology; progranulin; TAR DNA-binding protein 43; dystrophic neurites; autopsy; immunohistochemistry
22.  Human Postmortem Tissue: What Quality Markers Matter? 
Brain research  2006;1123(1):1-11.
Post mortem human brain tissue is used for the study of many different brain diseases. A key factor in conducting postmortem research is the quality of the tissue. Unlike animal tissue, whose condition at death can be controlled and influenced, human tissue can only be collected naturalistically. This introduces potential confounds, based both on pre- and postmortem conditions, that may influence the quality of tissue and its ability to yield accurate results. The traditionally recognized confounds that reduce tissue quality are agonal factors (e.g., coma, hypoxia, hyperpyrexia at the time of death), and long postmortem interval (PMI). We measured tissue quality parameters in over 100 postmortem cases collected from different sources and correlated them with RNA quality (as indicated by the RNA Integrity Number (RIN)) and with protein quality (as measured by the level of representative proteins). Our results show that the most sensible indicator of tissue quality is RIN and that there is a good correlation between RIN and the pH. No correlation developed between protein levels and the aforementioned factors. Moreover, even when RNA was degraded, the protein levels remained stable. However, these correlations did not prove true under all circumstances (e.g. thawed tissue, surgical tissue), that yielded unexpected quality indicators. These data also suggest that cases whose source was a Medical Examiner’s office represent high tissue quality.
doi:10.1016/j.brainres.2006.09.025
PMCID: PMC1995236  PMID: 17045977
brain; RIN; PMI; pH
23.  C9ORF72 repeat expansions in cases with previously identified pathogenic mutations 
Neurology  2013;81(15):1332-1341.
Objective:
To identify potential genetic modifiers contributing to the phenotypic variability that is detected in patients with repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), we investigated the frequency of these expansions in a cohort of 334 subjects previously found to carry mutations in genes known to be associated with a spectrum of neurodegenerative diseases.
Methods:
A 2-step protocol, with a fluorescent PCR and a repeat-primed PCR, was used to determine the presence of hexanucleotide expansions in C9ORF72. For one double mutant, we performed Southern blots to assess expansion sizes, and immunohistochemistry to characterize neuropathology.
Results:
We detected C9ORF72 repeat expansions in 4 of 334 subjects (1.2% [or 1.8% of 217 families]). All these subjects had behavioral phenotypes and also harbored well-known pathogenic mutations in either progranulin (GRN: p.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPT: p.P301L). Southern blotting of one double mutant with a p.C466LfsX46 GRN mutation demonstrated a long repeat expansion in brain (>3,000 repeats), and immunohistochemistry showed mixed neuropathology with characteristics of both C9ORF72 expansions and GRN mutations.
Conclusions:
Our findings indicate that co-occurrence of 2 evidently pathogenic mutations could contribute to the pleiotropy that is detected in patients with C9ORF72 repeat expansions. These findings suggest that patients with known mutations should not be excluded from further studies, and that genetic counselors should be aware of this phenomenon when advising patients and their family members.
doi:10.1212/WNL.0b013e3182a8250c
PMCID: PMC3806926  PMID: 24027057
24.  Globular glial tauopathies (GGT): consensus recommendations 
Acta neuropathologica  2013;126(4):537-544.
Rrecent studies have highlighted a group of 4-repeat (4R) tauopathies that are characterised neuropathologically by widespread, globular glial inclusions (GGIs). Tau immunohistochemistry reveals 4R immunore-active globular oligodendroglial and astrocytic inclusions and the latter are predominantly negative for Gallyas silver staining. These cases are associated with a range of clinical presentations, which correlate with the severity and distribution of underlying tau pathology and neurodegeneration. Their heterogeneous clinicopathological features combined with their rarity and under-recognition have led to cases characterised by GGIs being described in the literature using various and redundant terminologies. In this report, a group of neuropathologists form a consensus on the terminology and classification of cases with GGIs. After studying microscopic images from previously reported cases with suspected GGIs (n = 22), this panel of neuropathologists with extensive experience in the diagnosis of neurodegenerative diseases and a documented record of previous experience with at least one case with GGIs, agreed that (1) GGIs were present in all the cases reviewed; (2) the morphology of globular astrocytic inclusions was different to tufted astrocytes and finally that (3) the cases represented a number of different neuropathological subtypes. They also agreed that the different morphological subtypes are likely to be part of a spectrum of a distinct disease entity, for which they recommend that the overarching term globular glial tauopathy (GGT) should be used. Type I cases typically present with frontotemporal dementia, which correlates with the fronto-temporal distribution of pathology. Type II cases are characterised by pyramidal features reflecting motor cortex involvement and corticospinal tract degeneration. Type III cases can present with a combination of frontotemporal dementia and motor neuron disease with fronto-temporal cortex, motor cortex and corticospinal tract being severely affected. extrapyramidal features can be present in Type II and III cases and significant degeneration of the white matter is a feature of all GGT subtypes. Improved detection and classification will be necessary for the establishment of neuropathological and clinical diagnostic research criteria in the future.
doi:10.1007/s00401-013-1171-0
PMCID: PMC3914659  PMID: 23995422
25.  2-hydroxyglutarate detection by magnetic resonance spectroscopy in IDH-mutated glioma patients 
Nature medicine  2012;18(4):624-629.
Mutations in isocitrate dehydrogenase 1 and 2 (IDH1, 2) have been demonstrated in the majority of World Health Organization grade 2 and grade 3 gliomas in adults. These mutations are associated with the accumulation of 2-hydroxyglutarate (2HG) within the tumor. Here we report the noninvasive detection of 2HG by proton magnetic resonance spectroscopy (MRS). The pulse sequence was developed and optimized with numerical and phantom analyses for 2HG detection. The concentrations of 2HG were estimated using spectral fitting in the tumors of 30 patients. Detection of 2HG correlated with mutations in IDH1 or IDH2 and with increased levels of D-2HG by mass spectrometry of resected tumor. Noninvasive detection of 2HG may prove to be a valuable diagnostic and prognostic biomarker.
doi:10.1038/nm.2682
PMCID: PMC3615719  PMID: 22281806

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