Georges Lévesque

HES1 is a novel interactor of the Fanconi anemia core complex

Fanconi anemia (FA) proteins are thought to play a role in chromosome stability and repair of DNA cross-links; however, these functions may not fully explain the developmental abnormalities and bone marrow failure that are characteristic of FA individuals. Here we associate the FA proteins with the Notch1 developmental pathway through a direct protein-protein interaction between the FA core complex and the hairy enhancer of split 1 (HES1). HES1 interaction with FA core complex members is dependent on a functional FA pathway. Cells depleted of HES1 exhibit an FA-like phenotype that includes cellular hypersensitivity to mitomycin C (MMC) and lack of FANCD2 monoubiquitination and foci formation. HES1 is also required for proper nuclear localization or stability of some members of the core complex. Our results suggest that HES1 is a novel interacting protein of the FA core complex.

Presenilin-1 interacts directly with the β-site amyloid protein precursor cleaving enzyme (BACE1)

A neuropathological hallmark of Alzheimers disease is the presence of amyloid plaques. The major constituent of these plaques, occurring largely in brain areas important for memory and cognition, is the 40-42 amyloid residues (Abeta). Abeta is derived from the amyloid protein precursor after cleavage by the recently identified beta-secretase (BACE1) and the putative gamma-secretase complex containing presenilin 1 (PS1). In an attempt to develop a functional secretase enzymatic assay in yeast we demonstrate a direct binding between BACE1 and PS1. This interaction was confirmed in vivo using coimmunoprecipitation and colocalization studies in human cultured cells. Our results show that PS1 preferably binds immature BACE1, thus possibly acting as a functional regulator of BACE1 maturation and/or activity.

Hypothermia-induced hyperphosphorylation: a new model to study tau kinase inhibitors

Tau hyperphosphorylation is one hallmark of Alzheimers disease (AD) pathology. Pharmaceutical companies have thus developed kinase inhibitors aiming to reduce tau hyperphosphorylation. One obstacle in screening for tau kinase inhibitors is the low phosphorylation levels of AD-related phospho-epitopes in normal adult mice and cultured cells. We have shown that hypothermia induces tau hyperphosphorylation in vitro and in vivo. Here, we hypothesized that hypothermia could be used to assess tau kinase inhibitors efficacy. Hypothermia applied to models of biological gradual complexity such as neuronal-like cells, ex vivo brain slices and adult non-transgenic mice leads to tau hyperphosphorylation at multiple AD-related phospho-epitopes. We show that Glycogen Synthase Kinase-3 inhibitors LiCl and AR-A014418, as well as roscovitine, a cyclin-dependent kinase 5 inhibitor, decrease hypothermia-induced tau hyperphosphorylation, leading to different tau phosphorylation profiles. Therefore, we propose hypothermia-induced hyperphosphorylation as a reliable, fast, convenient and inexpensive tool to screen for tau kinase inhibitors.

δ-Catenin is genetically and biologically associated with cortical cataract and future Alzheimer-related structural and functional brain changes.

Multiple lines of evidence suggest that specific subtypes of age-related cataract (ARC) and Alzheimer disease (AD) are related etiologically. To identify shared genetic factors for ARC and AD, we estimated co-heritability of quantitative measures of cataract subtypes with AD-related brain MRI traits among 1,249 members of the Framingham Eye Study who had a brain MRI scan approximately ten years after the eye exam. Cortical cataract (CC) was found to be co-heritable with future development of AD and with several MRI traits, especially temporal horn volume (THV, ρ = 0.24, P<10−4). A genome-wide association study using 187,657 single nucleotide polymorphisms (SNPs) for the bivariate outcome of CC and THV identified genome-wide significant association with CTNND2 SNPs rs17183619, rs13155993 and rs13170756 (P<2.6×10−7). These SNPs were also significantly associated with bivariate outcomes of CC and scores on several highly heritable neuropsychological tests (5.7×10−9≤P<3.7×10−6). Statistical interaction was demonstrated between rs17183619 and APP SNP rs2096488 on CC (P = 0.0015) and CC-THV (P = 0.038). A rare CTNND2 missense mutation (G810R) 249 base pairs from rs17183619 altered δ-catenin localization and increased secreted amyloid-β1–42 in neuronal cell culture. Immunohistopathological analysis of lens tissue obtained from two autopsy-confirmed AD subjects and two non-AD controls revealed elevated expression of δ-catenin in epithelial and cortical regions of lenses from AD subjects compared to controls. Our findings suggest that genetic variation in delta catenin may underlie both cortical lens opacities in mid-life and subsequent MRI and cognitive changes that presage the development of AD.

Hyperinsulinemia and Abdominal Obesity Affect the Expression of Hypertriglyceridemia in Heterozygous Familial Lipoprotein Lipase Deficiency

We have reported three missense mutations (G188E, P207L, and D250N) in the lipoprotein lipase (LPL) gene among French-Canadians, resulting in the absence of measurable postheparin plasma LPL activity in homozygotes. Presence of triglyceride- and cholesterolrich VLDL, as well as cholesterol-poor HDL particles, has been shown in heterozygotes affected by partial reduction in postheparin LPL activity. However, significant heterogeneity in their plasma triglyceride levels has been found, even among individuals carrying the same LPL gene mutation, indicating that factors other than LPL deficiency could affect the phenotypic expression of hypertriglyceridemia in the heterozygous state. The aim of the present study was to examine the combined effects of abdominal fat accumulation and hyperinsulinemia on plasma triglyceride levels among heterozygous patients for familial LPL deficiency. Based on sex and BMI, 43 heterozygotes (25 women and 18 men) were matched with noncarrier control subjects. Our data indicate that heterozygotes with higher abdominal fat deposition, as defined as waist girth values above the 50th percentile, had higher plasma triglyceride levels than nonobese heterozygotes. However, an important proportion of male heterozygote subjects were hypertriglyceridemic, even in absence of abdominal obesity, suggesting that another factor(s) was involved in the modulation of hypertriglyceridemia in these subjects. Indeed, multivariate analyses revealed that fasting hyperinsulinemia was a significant correlate of hypertriglyceridemia among these heterozygotes. Results of the present study indicate that abdominal obesity and hyperinsulinemia both have deleterious effects on plasma triglyceride levels in familial LPL deficiency. It is suggested that heterozygotes with moderate obesity and/or insulin resistance may be at higher risk of coronary artery disease because of the expression of an atherogenic lipoprotein phenotype among these patients.

DETECTION OF THE PERIPHERAL NERVOUS SYSTEM (PNS)-TYPE GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) AND ITS MRNA IN HUMAN LYMPHOCYTES

Glial fibrillary acidic protein (GFAP), an astroglial marker, has been detected in the peripheral nervous system (PNS) in a shorter version and its mRNA in a longer form (β‐type) than the brain α‐type. To determine the characteristics of the GFAP gene expression in nonneural cells, we have investigated its in vivo transcription and translation products in human lymphocytes. Using RT‐PCR, we demonstrate that the GFAP gene is transcribed in these cells. Most or all of the mRNA resulting from this transcription was longer than the brain‐type at its 5′ end and thus may correspond to the β‐type. In addition, immunoblotting of lymphocyte extracts with a monoclonal antibody revealed a 41 KDa fragment instead of the 50 KDa expected from brain GFAP. These results suggest that GFAP expression in lymphocytes is preferentially of the PNS β‐type giving rise to longer mRNA and shorter protein. However, compared to two other astroglial mRNAs (S‐100β and aldolase C) which were synthesized in significant amounts in lymphocytes, GFAP mRNA was detected in minute amounts representing 0.03% of the brain level. This low expression may subserve a special role in lymphocytes since it is translated. J. Neurosci. Res. 48: 53–62, 1997.

Oligomerization of human presenilin‐1 fragments

To gain insight into presenilin‐1 (PS1) structural aspects, we explored the structure–function relationship of its N‐ and C‐terminal (NTF and CTF, respectively) complexes. We demonstrated that both NTF and CTF act as independent but inter‐changing binding units capable of binding each other (NTF/CTF) or their homologues (NTF/NTF; CTF/CTF). The Alzheimers disease‐associated PS1 mutations Y115H and M146L do not affect their ability to hetero‐ and/or homodimerize, thus conserving their basic integrity and function(s). These results suggest that PS1 associates intra‐molecularly to form higher order complexes, which may be needed for endoproteolytic cleavage and/or γ‐secretase‐associated activity.

The Fanconi Anemia Core Complex Acts as a Transcriptional Co-regulator in Hairy Enhancer of Split 1 Signaling

Mutations in one of the 13 Fanconi anemia (FA) genes cause a progressive bone marrow failure disorder associated with developmental abnormalities and a predisposition to cancer. Although FA has been defined as a DNA repair disease based on the hypersensitivity of patient cells to DNA cross-linking agents, FA patients develop various developmental defects such as skeletal abnormalities, microphthalmia, and endocrine abnormalities that may be linked to transcriptional defects. Recently, we reported that the FA core complex interacts with the transcriptional repressor Hairy Enhancer of Split 1 (HES1) suggesting that the core complex plays a role in transcription. Here we show that the FA core complex contributes to transcriptional regulation of HES1-responsive genes, including HES1 and the cyclin-dependent kinase inhibitor p21cip1/waf1. Chromatin immunoprecipitation studies show that the FA core complex interacts with the HES1 promoter but not the p21cip1/waf1 promoter. Furthermore, we show that the FA core complex interferes with HES1 binding to the co-repressor transducin-like-Enhancer of Split, suggesting that the core complex affects transcription both directly and indirectly. Taken together these data suggest a novel function of the FA core complex in transcriptional regulation.

The Fanconi anemia pathway has a dual function in Dickkopf-1 transcriptional repression

Significance Fanconi anemia (FA) is a devastating disease associated with a progressive bone marrow failure (BMF) and clonal proliferation of primitive hematopoietic cells that leads to leukemia. In an effort to understand the molecular basis of BMF and leukemogenesis in FA, we recently uncovered a unique function of proteins associated with FA in transcriptional regulation that translates into elevated levels of the signaling molecule Dickkopf-1 (DKK1). Overproduction of DKK1 has been shown to alter functions in hematopoiesis and to promote hematologic malignancies. Thus, our findings represent a crucial step in the development of strategies aimed at preventing BMF and/or clonal hematopoiesis in patients with FA. Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with a progressive decline in hematopoietic stem cells, developmental defects, and predisposition to cancer. These various phenotypic features imply a role of FA proteins in molecular events regulating cellular homeostasis. Interestingly, we previously found that the Fanconi C protein (FANCC) interacts with the C-terminal-binding protein-1 (CtBP1) involved in transcriptional regulation. Here we report that FANCC with CtBP1 forms a complex with β-catenin, and that β-catenin activation through glycogen synthase kinase 3β inhibition leads to FANCC nuclear accumulation and FA pathway activation, as measured by the Fanconi D2 protein (FANCD2) monoubiquitination. β-catenin and FANCC nuclear entry is defective in FA mutant cells and in cells depleted of the Fanconi A protein or FANCD2, suggesting that integrity of the FA pathway is required for FANCC nuclear activity. We also report that FANCC with CtBP1 acts as a negative regulator of Dickkopf-1 (DKK1) expression, and that a FA disease-causing mutation in FANCC abrogates this function. Our findings reveal that a defective FA pathway leads to up-regulation of DKK1, a molecule involved in hematopoietic malignancies.

A Nuclear Function for the Presenilin 1 Neuronal Partner NPRAP/δ-Catenin

Presenilin-1 (PS1) is a broadly expressed transmembrane protein that is often mutated in familial Alzheimers disease (AD). In addition to its role in amyloid production, PS1 interacts with several protein partners, including the neural plakophilin-related armadillo protein (NPRAP or δ-catenin). Although studies have suggested that NPRAP affects cell adhesion, other data suggest that it can modulate gene expression. To investigate the transcriptional effects of NPRAP, we over-expressed NPRAP and measured gene expression using a microarray. We found that multiple genes, including BCHE, which has been linked to AD, were regulated by NPRAP. Furthermore, we showed that NPRAP nuclear translocation was required for gene regulation. Our results implicate NPRAP as a brain-specific signaling molecule with distinct roles at the cell junction and the nucleus.