Maureen E. Balestra

Rosiglitazone increases dendritic spine density and rescues spine loss caused by apolipoprotein E4 in primary cortical neurons

Convergent evidence has revealed an association between insulin resistance and Alzheimers disease (AD), and the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, an insulin sensitizer and mitochondrial activator, improves cognition in patients with early or mild-to-moderate AD. Apolipoprotein (apo) E4, a major genetic risk factor for AD, exerts neuropathological effects through multiple pathways, including impairment of dendritic spine structure and mitochondrial function. Here we show that rosiglitazone significantly increased dendritic spine density in a dose-dependent manner in cultured primary cortical rat neurons. This effect was abolished by the PPAR-γ-specific antagonist, GW9662, suggesting that rosiglitazone exerts this effect by activating the PPAR-γ pathway. Furthermore, the C-terminal-truncated fragment of apoE4 significantly decreased dendritic spine density. Rosiglitazone rescued this detrimental effect. Thus, rosiglitazone might improve cognition in AD patients by increasing dendritic spine density.

Genetic Correction of Tauopathy Phenotypes in Neurons Derived from Human Induced Pluripotent Stem Cells

Summary Tauopathies represent a group of neurodegenerative disorders characterized by the accumulation of pathological TAU protein in brains. We report a human neuronal model of tauopathy derived from induced pluripotent stem cells (iPSCs) carrying a TAU-A152T mutation. Using zinc-finger nuclease-mediated gene editing, we generated two isogenic iPSC lines: one with the mutation corrected, and another with the homozygous mutation engineered. The A152T mutation increased TAU fragmentation and phosphorylation, leading to neurodegeneration and especially axonal degeneration. These cellular phenotypes were consistent with those observed in a patient with TAU-A152T. Upon mutation correction, normal neuronal and axonal morphologies were restored, accompanied by decreases in TAU fragmentation and phosphorylation, whereas the severity of tauopathy was intensified in neurons with the homozygous mutation. These isogenic TAU-iPSC lines represent a critical advancement toward the accurate modeling and mechanistic study of tauopathies with human neurons and will be invaluable for drug-screening efforts and future cell-based therapies.

Intron-3 Retention/Splicing Controls Neuronal Expression of Apolipoprotein E in the CNS

Neuronal expression of apolipoprotein (apo) E4 may contribute to the pathogenesis of Alzheimers disease (AD). In studying how apoE expression is regulated in neurons, we identified a splicing variant of apoE mRNA with intron-3 retention (apoE-I3). ApoE-I3 mRNA was detected in neuronal cell lines and primary neurons, but not in astrocytic cell lines or primary astrocytes, from humans and mice by reverse transcription (RT)-PCR. In both wild-type and human apoE knock-in mice, apoE-I3 was found predominantly in cortical and hippocampal neurons by in situ hybridization. Cell fractionation and quantitative RT-PCR revealed that over 98% of the apoE-I3 mRNA was retained in the nucleus without protein translation. In transfected primary neurons, apoE expression increased dramatically when intron-3 was deleted from a genomic DNA construct and decreased markedly when intron-3 was inserted into a cDNA construct, suggesting that intron-3 retention/splicing controls apoE expression in neurons. In response to excitotoxic challenge, the apoE-I3 mRNA was markedly increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons in mice; apoE mRNA showed the opposite pattern. This apparent precursor–product relationship between apoE-I3 and apoE mRNA was supported by a transcriptional inhibition study. Thus, neuronal expression of apoE is controlled by transcription of apoE-I3 under normal conditions and by processing of apoE-I3 into mature apoE mRNA in response to injury.

Small Molecule Structure Correctors Abolish Detrimental Effects of Apolipoprotein E4 in Cultured Neurons

Background: Apolipoprotein E4 (apoE4), the major gene involved in Alzheimer disease, has a unique structure, intramolecular domain interaction, that is associated with neuropathology. Results: Potent small molecule structure correctors block apoE4 domain interaction and reverse apoE4 detrimental effects in cultured neurons. Conclusion: Structure correctors negate the detrimental effects of apoE4 in neurons. Significance: ApoE4 structure correctors could represent a therapeutic approach for treating apoE4-associated neuropathology. Apolipoprotein E4 (apoE4), the major genetic risk factor for late onset Alzheimer disease, assumes a pathological conformation, intramolecular domain interaction. ApoE4 domain interaction mediates the detrimental effects of apoE4, including decreased mitochondrial cytochrome c oxidase subunit 1 levels, reduced mitochondrial motility, and reduced neurite outgrowth in vitro. Mutant apoE4 (apoE4-R61T) lacks domain interaction, behaves like apoE3, and does not cause detrimental effects. To identify small molecules that inhibit domain interaction (i.e. structure correctors) and reverse the apoE4 detrimental effects, we established a high throughput cell-based FRET primary assay that determines apoE4 domain interaction and secondary cell- and function-based assays. Screening a ChemBridge library with the FRET assay identified CB9032258 (a phthalazinone derivative), which inhibits domain interaction in neuronal cells. In secondary functional assays, CB9032258 restored mitochondrial cytochrome c oxidase subunit 1 levels and rescued impairments of mitochondrial motility and neurite outgrowth in apoE4-expressing neuronal cells. These benefits were apoE4-specific and dose-dependent. Modifying CB9032258 yielded well defined structure-activity relationships and more active compounds with enhanced potencies in the FRET assay (IC50 of 23 and 116 nm, respectively). These compounds efficiently restored functional activities of apoE4-expressing cells in secondary assays. An EPR binding assay showed that the apoE4 structure correction resulted from direct interaction of a phthalazinone. With these data, a six-feature pharmacophore model was constructed for future drug design. Our results serve as a proof of concept that pharmacological intervention with apoE4 structure correctors negates apoE4 detrimental effects in neuronal cells and could be further developed as an Alzheimer disease therapeutic.

DGAT1 promoter polymorphism associated with alterations in body mass index, high density lipoprotein levels and blood pressure in Turkish women

Triglyceride synthesis is catalyzed by acyl CoA:diacylglycerol acyltransferases (DGAT), microsomal enzymes that use diacylglycerol and fatty acyl CoAs as substrates. Because DGAT1 expression is up‐regulated during adipocyte differentiation and DGAT1 deficiency is associated with leanness in mice, we hypothesized that alterations in DGAT1 expression may affect human body weight. We identified five polymorphisms in the human DGAT1 promoter and 5′ non‐coding sequence in a random Turkish population. Functional analysis of one common variant, C79T, revealed reduced promoter activity for the 79T allele in cultured cell lines. In 476 Turkish women, the 79T allele was associated with lower body mass index (BMI) (p = 0.004), conferring an odds ratio of 2.0 (95% CI = 1.30–3.07, p = 0.0001) for BMI ≤ 20. Interestingly, after controlling for the influence of BMI, the 79T allele was also associated with higher plasma HDL cholesterol levels (p = 0.0006) and lower diastolic blood pressure (p = 0.019) in these women. No association was found in Turkish men (n = 846). Our findings suggest that genetic variation at the DGAT1 locus may influence BMI and other metabolic parameters associated with cardiovascular risk in selected human populations.

Effect of Arginine 172 on the Binding of Apolipoprotein E to the Low Density Lipoprotein Receptor

The region of apolipoprotein E (apoE) that interacts directly with the low density lipoprotein (LDL) receptor lies in the vicinity of residues 136–150, where lysine and arginine residues are crucial for full binding activity. However, defective binding of carboxyl-terminal truncations of apoE3 has suggested that residues in the vicinity of 170–183 are also important. To characterize and define the role of this region in LDL receptor binding, we created either mutants of apoE in which this region was deleted or in which arginine residues within this region were sequentially changed to alanine. Deletion of residues 167–185 reduced binding activity (15% of apoE3), and elimination of arginines at positions 167, 172, 178, and 180 revealed that only position 172 affected binding activity (2% of apoE3). Substitution of lysine for Arg172 reduced binding activity to 6%, indicating a specific requirement for arginine at this position. The higher binding activity of the Δ167–185 mutant relative to the Arg172mutant (15% versus 2%) is explained by the fact that arginine residues at positions 189 and 191 are shifted in the deletion mutant into positions equivalent to 170 and 172 in the intact protein. Mutation of these residues and modeling the region around these residues suggested that the influence of Arg172 on receptor binding activity may be determined by its orientation at a lipid surface. Thus, the association of apoE with phospholipids allows Arg172 to interact directly with the LDL receptor or with other residues in apoE to promote its receptor-active conformation.

Structure-dependent Impairment of Intracellular Apolipoprotein E4 Trafficking and Its Detrimental Effects Are Rescued by Small-molecule Structure Correctors

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimer disease (AD) and likely contributes to neuropathology through various pathways. Here we report that the intracellular trafficking of apoE4 is impaired in Neuro-2a cells and primary neurons, as shown by measuring fluorescence recovery after photobleaching. In Neuro-2a cells, more apoE4 than apoE3 molecules remained immobilized in the endoplasmic reticulum (ER) and the Golgi apparatus, and the lateral motility of apoE4 was significantly lower in the Golgi apparatus (but not in the ER) than that of apoE3. Likewise, the immobile fraction was larger, and the lateral motility was lower for apoE4 than apoE3 in mouse primary hippocampal neurons. ApoE4 with the R61T mutation, which abolishes apoE4 domain interaction, was less immobilized, and its lateral motility was comparable with that of apoE3. The trafficking impairment of apoE4 was also rescued by disrupting domain interaction with the small-molecule structure correctors GIND25 and PH002. PH002 also rescued apoE4-induced impairments of neurite outgrowth in Neuro-2a cells and dendritic spine development in primary neurons. ApoE4 did not affect trafficking of amyloid precursor protein, another AD-related protein, through the secretory pathway. Thus, domain interaction renders more newly synthesized apoE4 molecules immobile and slows their trafficking along the secretory pathway. Correcting the pathological structure of apoE4 by disrupting domain interaction is a potential therapeutic approach to treat or prevent AD related to apoE4.

Two Distinct TATA-less Promoters Direct Tissue-specific Expression of the Rat Apo-B Editing Catalytic Polypeptide 1 Gene

The species and tissue specificity of apolipoprotein (apo) B mRNA editing is determined by the expression of apoB editing catalytic polypeptide 1 (APOBEC-1), the cytidine deaminase that catalyzes apoB mRNA editing. To understand the molecular mechanisms that regulate the transcription of APOBEC-1, we characterized rat APOBEC-1 cDNA and genomic DNA. cDNA cloning and RNase protection analysis showed two alternative promoters for the tissue-specific expression of APOBEC-1 in the liver and intestine, Pliv and Pint. Both promoters lack a TATA box, and Pint belongs to the MED-1 class of promoters, which initiate transcription at multiple sites. We also identified two allelic forms of the APOBEC-1 gene from the characterization of two rat APOBEC-1 P1 genomic clones, RE4 and RE5. The RE4 allele is 18 kilobases long and contains six exons and five introns, whereas the RE5 allele contains an additional ∼8 kilobases of intron sequences and an extra exon encoding a 5′-untranslated region; however, the APOBEC-1 transcripts from the two alleles appear to have similar, if not identical, functions. Transgenic mouse studies showed that Pliv was preferentially used in the liver, kidney, brain, and adipose tissues, whereas Pint was preferentially used in the small intestine, stomach, and lung. Our results suggest that the tissue-specific expression of APOBEC-1 is governed by multiple regulatory elements exerting control over a single coding sequence. The presence or absence of these regulatory elements may determine the tissue-specific expression of APOBEC-1 in other mammalian species.

Regulatable liver expression of the rabbit apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in mice lacking endogenous APOBEC-1 leads to aberrant hyperediting.

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which results in translation of the apoB-48 protein instead of the genomically encoded apoB-100. ApoB-48-containing lipoproteins are cleared more rapidly from plasma and are less atherogenic than apoB-100-containing low-density lipoproteins (LDLs). In humans, the intestine predominantly produces apoB-48 whereas the liver secretes apoB-100 only. To evaluate a potential therapeutic use for liver-induced apoB mRNA editing in humans, we investigated the efficiency and safety of transgenic expression of apoB mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) in the absence of endogenous editing in the mouse model. Here we show that regulatable tetO-mediated APOBEC-1 expression in the livers of gene-targeted mice lacking endogenous APOBEC-1 results in 30% apoB mRNA editing. In a time-course experiment, the expression of tetO-APOBEC-1 mRNA was suppressed within 2 days after mice were fed doxycycline and apoB mRNA editing and apoB-48 formation were suppressed within 4 days. However, tetO-APOBEC-1 expression resulted in regulatable aberrant hyperediting of several cytidines downstream of C(6666) in apoB mRNA and in novel APOBEC-1 target 1 (NAT1) mRNA. Several of the cytidines in apoB mRNA were hyperedited to a level similar to that of C(6666), although editing at C(6666) was lower than that in wild-type mice. These results demonstrate that even moderate APOBEC-1 expression can lead to hyperediting, limiting the single-gene approach for gene therapy with APOBEC-1.