Genome-wide association studies are identifying novel Alzheimer's disease (AD) risk factors. Elucidating the mechanism underlying these polymorphisms is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we elucidate the mechanism of action of the AD-associated polymorphism rs3865444 in the promoter of CD33, a member of the sialic acid-binding Ig-superfamily of lectins (SIGLECs). Immunostaining established that CD33 is expressed in microglia in human brain. Consistent with this finding, CD33 mRNA expression correlated well with expression of the microglial genes CD11b and AIF-1 and was modestly increased with AD status and the rs3865444C AD-risk allele. Analysis of CD33 isoforms identified a common isoform lacking exon 2 (D2-CD33). The proportion of CD33 expressed as D2-CD33 correlated robustly with rs3865444 genotype. Because rs3865444 is in the CD33 promoter region, we sought the functional polymorphism by sequencing CD33 from the promoter through exon 4. We identified a single polymorphism that is coinherited with rs3865444, i.e., rs12459419 in exon 2. Minigene RNA splicing studies in BV2 microglial cells established that rs12459419 is a functional single nucleotide polymorphism (SNP) that modulates exon 2 splicing efficiency. Thus, our primary findings are that CD33 is a microglial mRNA and that rs3865444 is a proxy SNP for rs12459419 that modulates CD33 exon 2 splicing. Exon 2 encodes the CD33 IgV domain that typically mediates sialic acid binding in SIGLEC family members. In summary, these results suggest a novel model wherein SNP-modulated RNA splicing modulates CD33 function and, thereby, AD risk.

Most, if not all, nociceptor sensory neurons are dependent on nerve growth factor (NGF) during early embryonic development. A large subpopulation of these sensory neurons loses NGF dependency between embryonic day 16 and postnatal day 14 and become responsive to glial cell line-derived growth factor (GDNF), a member of the transforming growth factor beta (TGF-beta) family. To examine the survival and phenotypic effects of GDNF on sensory neurons in vivo, we generated transgenic mice that overexpress GDNF in the skin. GDNF-overexpresser mice had increased numbers of small unmyelinated sensory neurons that express the tyrosine kinase receptor Ret and bind the plant isolectin B4 (IB4). Surprisingly, in wild-type and transgenic mice, few ( approximately 2%) IB4-positive neurons expressed the vanilloid receptor VR1, a heat-sensitive receptor expressed by many IB4-positive neurons of the rat. Thus, in mouse, GDNF-dependent IB4-positive neurons must use a non-VR1 heat receptor. In addition, the behavior of GDNF-overexpresser animals to noxious heat or mechanical stimuli was indistinguishable from wild-type animals, indicating that, on a behavioral level, peripherally applied GDNF does not alter the sensitivity of the somatosensory system.

Single nucleotide polymorphisms (SNPs) that alter exon splicing efficiency are an emerging class of functional genetic variants. Since mutations in low-density lipoprotein receptor (LDLR) are a primary cause of familial hypercholesterolemia, we evaluated whether LDLR SNPs may alter splicing efficiency and cholesterol homeostasis. A SNP within LDLR exon 12, rs688, was identified in silico as neutralizing a putative exon splicing enhancer. Studies in human liver samples established that this SNP was associated with significantly decreased LDLR exon 12 splicing efficiency in women in vivo. In vitro minigene splicing studies qualitatively replicated these in vivo results and demonstrated that rs688 specifically modulates splicing efficiency. These effects on splicing may be physiologically relevant because the presence of the rs688 minor allele associates with increased total and LDL-cholesterol in female members of the Framingham Offspring Study. The largest rs688-associated cholesterol differences were observed in pre-menopausal women. In summary, these studies identify an LDLR SNP present in approximately 60% of Caucasians that is associated with significant 10% increases in total and LDL-cholesterol in pre-menopausal women.

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and related this to altered mitochondrial DNA replication found in vivo. 5 We explored enzymologic events in the interaction of purified DNA pol-y with AZTTP. Toxic mechanisms in AZT mitochondrial myopathy may be analogous in some ways to pharmacologic mechanisms in the antiretroviral action of AZT.2 The present study showed that AZTTP inhibited DNA pol-y with mixed inhibition kinetics. AZTTP acted both as an alternate substrate for dTTP with DNA pol-y and competed with dTTP for DNA pol-y nucleotide binding (as seen with AZTTP and some cellular polymerases). Materials and Methods MaterialsReagents were analytic grade I. AZTTP was from Moravek Biochemicals, Brea, Calif. [3H]dTTP was from Amersham. All enzyme inhibitor assays were replicated from five to eight times, and each assay point was performed in triplicate within each run. Calculated arithmetic means from the triplicate assays were used to plot kinetic data. Mitochondrial Isolation ProceduresFor mitochondrial isolation, all procedures took place either on ice or at 4°C and resembled those used by us and by others in the past.78 Fresh bovine hearts were obtained from the slaughterhouse (courtesy of Kluener Packing Co, Cincinnati, Ohio) approximately 20 minutes before beginning the preparation. Tissue was minced into 1-cm cubes and homogenized in buffer M that consisted of (mmol/L) sucrose, 250; Tris-HCI, 50 (pH 8.0); 2-mercaptoethanol (2-ME), 5; EDTA, 1; MgCl2, 5; and KCI, 25. Buffer volume was increased 10-fold, and the mixture was homogenized with a hand-held Potter-Elvejhem homogenizer (Fisher Scientific, Pittsburgh, Pa). Debris was pelleted at 500g. The mitochondria were pelleted from the supernatant solution by centrifugation at 8500g for 10 minutes.