Mirta Mihovilovic

The cis-regulatory effect of an Alzheimer’s disease-associated poly-T locus on expression of TOMM40 and apolipoprotein E genes

We investigated the genomic region spanning the Translocase of the Outer Mitochondrial Membrane 40‐kD (TOMM40) and Apolipoprotein E (APOE) genes, that has been associated with the risk and age of onset of late‐onset Alzheimers disease (LOAD) to determine whether a highly polymorphic, intronic poly‐T within this region (rs10524523; hereafter, 523) affects expression of the APOE and TOMM40 genes. Alleles of this locus are classified as S, short; L, long; and VL, very long based on the number of T residues.

Thymocytes and cultured thymic epithelial cells express transcripts encoding α-3, α-5 and β-4 subunits of neuronal nicotinic acetylcholine receptors: preferential transcription of the α-3 and β-4 genes by immature CD4+8+ thymocytes

Thymic tissues express transcripts encoding the alpha-3, alpha-5 and beta-4 subunits of nicotinic neuronal acetylcholine receptors (AcChRs) suggesting that neuronal AcChRs similar to those expressed in ganglia are expressed in the thymus. Transcription occurs in both isolated thymocytes and thymic epithelial cells. RT-PCR analyses of thymocyte subsets indicate that immature CD4 + 8 + thymocytes express higher levels of the alpha-3 and beta-4 transcripts than more mature thymocytes. Compared to freshly isolated thymocytes, peripheral blood lymphocytes do not express alpha-3 and beta-4 AcChR subunit transcripts. Cultured thymocytes rapidly down-regulate transcription of the alpha-3 and beta-4 AcChR subunit genes by a process that is not reversed by stimulation with phytohemagglutinin and IL-2. Thus our results indicate that there is transcriptional regulation of neuronal AcChR subunit genes during the process of thymocyte maturation and that factors within the thymic microenvironment influence expression of the alpha-3 and beta-4 AcChR subunit genes by developing T cells.

Expression of mRNAs in human thymus coding for the α3 subunit of a neuronal acetylcholine receptor

We report the isolation of clones from a human thymus cDNA library that code for the alpha 3 subunit of a neuronal acetylcholine receptor (AcChR). The clones hybridize to one major 3.0-kb mRNA thymic species and four minor ones of approximately 2.3, 4.0, 5.0, and 6.5 kb, but they do not hybridize to human muscle mRNA. These clones may be of value in defining the cholinergic thymic makeup and the putative role that a thymic AcChR may have in the triggering and/or maintenance of an anti-AcChR response in the autoimmune condition myasthenia gravis.

APOE4-VLDL Inhibits the HDL-Activated Phosphatidylinositol 3-Kinase/Akt Pathway via the Phosphoinositol Phosphatase SHIP2

Endothelial cell dysfunction and apoptosis are critical in the pathogenesis of atherosclerotic cardiovascular disease (CVD). Both endothelial cell apoptosis and atherosclerosis are reduced by high-density lipoprotein (HDL). Low HDL levels increase the risk of CVD and are also a key characteristic of the metabolic syndrome. The apolipoprotein E4 (APOE4) allele also increases the risk of atherosclerosis and CVD. We previously demonstrated that the antiapoptotic activity of HDL is inhibited by APOE4 very-low-density lipoprotein (APOE4-VLDL) in endothelial cells, an effect similar to reducing the levels of HDL. Here we establish the intracellular mechanism by which APOE4-VLDL inhibits the antiapoptotic pathway activated by HDL. We show that APOE4-VLDL diminishes the phosphorylation of Akt by HDL but does not alter phosphorylation of c-Jun N-terminal kinase, p38, or Src family kinases by HDL. Furthermore APOE4-VLDL inhibits Akt phosphorylation by reducing the phosphatidylinositol 3-kinase product phosphatidylinositol-(3,4,5)-triphosphate (PI[3,4,5]P3). We further demonstrate that APOE4-VLDL reduces PI(3,4,5)P3, through the phosphoinositol phosphatase SHIP2, and not through PTEN. SHIP2 is already implicated as an independent risk factor for type II diabetes, hypertension and obesity, which are also all components of the metabolic syndrome and independent risk factors for CVD. Significantly, the association between CVD and type 2 diabetes or hypertension is further increased by the APOE4 allele. Therefore the activation of SHIP2 by APOE4-VLDL, with the subsequent inhibition of the HDL/Akt pathway, is a novel and significant biological mechanism and may be a critical intermediate by which APOE4 increases the risk of atherosclerotic CVD.

Human × Human Hybridomas from Patients with Myasthenia Gravis: Possible Tools for Idiotypic Therapy for Myastheniaa

Hybridomas secreting monoclonal antibodies directed against the nicotinic acetylcholine receptor have been developed from rats with experimental autoimmune myasthenia gravis and from a patient with myasthenia gravis. Rat monoclonal antibodies were characterized by their ability to bind to electroblotted acetylcholine receptor subunits. Of 34 tested, 22 bound to the alpha subunit. Three bound to other subunits, and the remainder appeared to bind only to the native molecule. The human monoclonal antibodies were analyzed with respect to their binding to membrane-bound and solubilized acetylcholine receptor. Many bound with greater affinity to the membrane-bound form of the antigen. Two rat monoclonal antibodies capable of passively transferring experimental autoimmune myasthenia gravis, and with reactivities to the alpha subunit of the acetylcholine receptor, were employed to produce isogeneic monoclonal antiidiotypic antibodies. When they were injected prior to immunization with acetylcholine receptor, two of the antiidiotypic antibodies directed against framework determinants prevented the development of experimental autoimmune myasthenia gravis. This observation raises the possibility that the human monoclonal antibodies will be useful in the development of idiotypic treatment of the human disease.

The Alu neurodegeneration hypothesis: A primate-specific mechanism for neuronal transcription noise, mitochondrial dysfunction, and manifestation of neurodegenerative disease

It is hypothesized that retrotransposons have played a fundamental role in primate evolution and that enhanced neurologic retrotransposon activity in humans may underlie the origin of higher cognitive function. As a potential consequence of this enhanced activity, it is likely that neurons are susceptible to deleterious retrotransposon pathways that can disrupt mitochondrial function. An example is observed in the TOMM40 gene, encoding a β‐barrel protein critical for mitochondrial preprotein transport. Primate‐specific Alu retrotransposons have repeatedly inserted into TOMM40 introns, and at least one variant associated with late‐onset Alzheimers disease originated from an Alu insertion event. We provide evidence of enriched Alu content in mitochondrial genes and postulate that Alus can disrupt mitochondrial populations in neurons, thereby setting the stage for progressive neurologic dysfunction. This Alu neurodegeneration hypothesis is compatible with decades of research and offers a plausible mechanism for the disruption of neuronal mitochondrial homeostasis, ultimately cascading into neurodegenerative disease.

Apolipoprotein E genotype as a determinant of survival in chronic lymphocytic leukemia

Survival of chronic lymphocytic leukemia (CLL) cells requires sustained activation of the antiapoptotic PI-3-K/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or antiapoptotic. High-density lipoprotein particles are antiapoptotic through sphingosine-1-phosphate receptor 3-mediated activation of the PI-3-K/Akt pathway. Apolipoprotein E4 (apoE4)–very low density lipoproteins (VLDL) increase apoptosis, but the apoE2-VLDL and apoE3-VLDL isoforms do not. As increased B-cell apoptosis favors longer survival of CLL patients, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. We report here that women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients. VLDL is metabolized to low-density lipoprotein through lipoprotein lipase. Higher levels of lipoprotein lipase mRNA in these CLL patients correlated with shorter survival. The beneficial effect of APOE4 in CLL survival is likely mediated through APOE4 allele-specific regulation of leukemia cell apoptosis. The APOE allele and genotype distribution in these CLL patients is the same as in unaffected control populations, suggesting that although APOE genotype influences CLL outcome and response to therapy, it does not alter susceptibility to developing this disease.

High-fat/high-cholesterol diet promotes a S1P receptor-mediated antiapoptotic activity for VLDL

Withdrawing growth factors or serum from endothelial cells leads to the activation of effector caspases 3 and 7, resulting in apoptotic cell death. HDL protects against caspase induction through sphingosine-1-phosphate (S1P) receptors. This anti-caspase activity of HDL is antagonized by VLDL from apolipoprotein E4 (apoE4) (genotype, APOE4/4; apolipoprotein, apoE) targeted replacement (TR) mice, but not by VLDL from TR APOE3/3 mice, and requires the binding of apoE4-VLDL to an LDL receptor family member. In the absence of HDL, apoE4-VLDL and apoE3-VLDL from TR mice have limited antiapoptotic activity. In contrast, we show here that a high-fat/high-cholesterol/cholate diet (HFD) radically alters this biological activity of VLDL. On HFD, both apoE3-VLDL and apoE4-VLDL (HFD VLDL) inhibit caspase 3/7 activation initiated by serum withdrawal. This activity of HFD VLDL is independent of an LDL receptor family member but requires the activation of S1P3 receptors, as shown by the ability of pharmacological block of S1P receptors by VPC 23019 and by small interfering RNA-mediated downregulation of S1P3 receptors to inhibit HFD VLDL anticaspase activity.

The Role of Upregulated APOE in Alzheimer’s Disease Etiology

The first and most firmly established genetic risk factor for sporadic late onset Alzheimer’s disease (LOAD) is the e4 allele of the apolipoprotein E (APOE) gene [1]. Carrying the APOEe4 variant significantly increases the lifetime risk for LOAD, with the number of copies present indicative of level of risk [1,2] and is associated with lower age of clinical disease onset [1,3–6]. Furthermore, genome-wide association studies (GWAS) for sporadic LOAD confirmed that APOE is the major susceptibility genomic region for the disease and reported significant associations with markers within the APOE linkage disequilibrium (LD) locus (contains APOE, TOMM40 and APOC1 genes). The strongest association signal (by wide margin) in these studies was found at the APOE LD region and no other LOAD-association in the human genome remotely approached the same level of significance [7–10]. However, the molecular mechanism underlying the reported genetic LOAD-associations with APOE LD region in general and APOEe4 haplotype in particular has yet to be discovered.

CONSEQUENCES OF INCREASED TOMM40 EXPRESSION ON CELLULAR ENERGETICS

neflamapimod for inhibition of cytokine production (Duffy, 2011), and correlates well with dose/concentration-response seen for episodic memory in the human clinical studies. Conclusions: The findings provide the first evidence that p38a antagonism could be a means to therapeutically target Alzheimer-related endosomal dysfunction. Further, the correlation between in vitro potency of neflamapomid on reversing endolysosomal dysfunction with clinical dose-response suggests that this pharmacological effect, rather than anti-inflammatory activity, may underlie the clinical activity of neflamapimod on episodic memory.