Faculty Bibliography

BACKGROUND: Alcadeinalpha (Alcalpha) is a neuronal membrane protein that colocalizes with the Alzheimer's amyloid-beta precursor protein (APP). Successive cleavage of APP by beta- and gamma-secretases generates the aggregatable amyloid-beta peptide (Abeta), while cleavage of APP or Alcalpha by alpha- and gamma-secretases generates non-aggregatable p3 or p3-Alcalpha peptides. Abeta and p3-Alcalpha can be recovered from human cerebrospinal fluid (CSF). We have previously reported alternative processing of APP and Alcalpha in the CSF of some patients with sporadic mild cognitive impairment (MCI) and AD (SAD). RESULTS: Using the sandwich enzyme-linked immunosorbent assay (ELISA) system that detects total p3-Alcalpha, we determined levels of total p3-Alcalpha in CSF from subjects in one of four diagnostic categories (elderly controls, MCI, SAD, or other neurological disease) derived from three independent cohorts. Levels of Abeta40 correlated with levels of total p3-Alcalpha in all cohorts. CONCLUSIONS: We confirm that Abeta40 is the most abundant Abeta species, and we propose a model in which CSF p3-Alcalpha can serve as a either (1) a nonaggregatable surrogate marker for gamma-secretase activity; (2) as a marker for clearance of transmembrane domain peptides derived from integral protein catabolism; or (3) both. We propose the specification of an MCI/SAD endophenotype characterized by co-elevation of levels of both CSF p3-Alcalpha and Abeta40, and we propose that subjects in this category might be especially responsive to therapeutics aimed at modulation of gamma-secretase function and/or transmembrane domain peptide clearance. These peptides may also be used to monitor the efficacy of therapeutics that target these steps in Abeta metabolism.

The proliferation/differentiation balance of stem and progenitor cell populations must respond to the physiological needs of the organism [1, 2]. Mechanisms underlying this plasticity are not well understood. The C. elegans germline provides a tractable system to study the influence of the environment on progenitor cells (stem cells and their proliferative progeny). Germline progenitors accumulate during larval stages to form an adult pool from which gametes are produced. Notch pathway signaling from the distal tip cell (DTC) niche to the germline maintains the progenitor pool [3-5], and the larval germline cell cycle is boosted by insulin/IGF-like receptor signaling [6]. Here we show that, independent of its role in the dauer decision, TGF-beta regulates the balance of proliferation versus differentiation in the C. elegans germline in response to sensory cues that report population density and food abundance. Ciliated ASI sensory neurons are required for TGF-beta-mediated expansion of the larval germline progenitor pool, and the TGF-beta receptor pathway acts in the germline stem cell niche. TGF-beta signaling thereby couples germline development to the quality of the environment, providing a novel cellular and molecular mechanism linking sensory experience of the environment to reproduction.

Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Delta4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Delta4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Delta4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Delta4-SHIVKU2 and CAL-Delta4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-gamma ELISPOT responses in Delta4-SHIVKU2- and CAL-Delta4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

Damage to the hippocampal formation results in a profound temporally graded retrograde amnesia, implying that it is necessary for memory acquisition but not its long-term storage. It is therefore thought that memories are transferred from the hippocampus to the cortex for long-term storage in a process called systems consolidation (Dudai and Morris, 2000). Where in the cortex this occurs remains an open question. Recent work (Frankland et al., 2005; Vetere et al., 2011) suggests the anterior cingulate cortex (ACC) as a likely candidate area, but there is little direct electrophysiological evidence to support this claim. Previously, we demonstrated object-associated firing correlates in caudal ACC during tests of recognition memory and described evidence of neuronal responses to where an object had been following a brief delay. However, long-term memory requires evidence of more durable representations. Here we examined the activity of ACC neurons while testing for long-term memory of an absent object. Mice explored two objects in an arena and then were returned 6 h later with one of the objects removed. Mice continued to explore where the object had been, demonstrating memory for that object. Remarkably, some ACC neurons continued to respond where the object had been, while others developed new responses in the absent object's location. The incidence of absent-object responses by ACC neurons was greatly increased with increased familiarization to the objects, and such responses were still evident 1 month later. These data strongly suggest that the ACC contains neural correlates of consolidated object/place association memory.

Clathrin and the epithelial-specific clathrin adaptor AP-1B mediate basolateral trafficking in epithelia. However, several epithelia lack AP-1B, and mice knocked out for AP-1B are viable, suggesting the existence of additional mechanisms that control basolateral polarity. Here, we demonstrate a distinct role of the ubiquitous clathrin adaptor AP-1A in basolateral protein sorting. Knockdown of AP-1A causes missorting of basolateral proteins in MDCK cells, but only after knockdown of AP-1B, suggesting that AP-1B can compensate for lack of AP-1A. AP-1A localizes predominantly to the TGN, and its knockdown promotes spillover of basolateral proteins into common recycling endosomes, the site of function of AP-1B, suggesting complementary roles of both adaptors in basolateral sorting. Yeast two-hybrid assays detect interactions between the basolateral signal of transferrin receptor and the medium subunits of both AP-1A and AP-1B. The basolateral sorting function of AP-1A reported here establishes AP-1 as a major regulator of epithelial polarity.

High residual platelet reactivity (HRPR) on clopidogrel is a predictor of recurrent ischemic events in patients undergoing percutaneous coronary interventions (PCI). Significant intraindividual variability in platelet aggregation on repeat testing has been reported. To understand factors contributing to the variability in platelet aggregation testing, we examined clinical and laboratory elements linked to HRPR in 255 consecutive patients tested ≥12 hours after PCI using light transmission aggregometry (LTA) in response to adenosine diphosphate 5 μmol/L and VerifyNow P2Y12 assay (VNP2Y12; Accumetrics). HRPR was defined as >46% residual aggregation for LTA and >236 P2Y12 response units (PRUs) for VNP2Y12. On multivariate analysis the only variable independently associated with HRPR with both LTA and VNP2Y12 was laboratory-defined anemia. Prevalences of HRPR by LTA were 34.3% in anemic patients, 15.6% in patients with normal hemoglobin levels, and 59.8% versus 25.9% by VNP2Y12 (p <0.005 for the 2 comparisons). In a subgroup of 50 patients, testing was done before and after the clopidogrel loading dose. At baseline there were no differences in platelet aggregation with either assay; however, absolute decrease in reactivity after the clopidogrel load was significantly less in anemic patients compared to patients with normal hemoglobin (change in residual aggregation by LTA 15.8 ± 5.8% vs 28.8 ± 3.2%, p <0.05; change in PRU by VNP2Y12 56.5 ± 35.5 vs 145.0 ± 14.2 PRUs, p <0.05, respectively). In conclusion, anemia is an important contributor to apparent HRPR on clopidogrel and may explain some of the intraindividual variability of platelet aggregation testing.

Women carriers of mutations in the tumor suppressor gene, BRCA1, have an up to 80% risk of developing breast cancer, as well as a 40% risk of ovarian cancer. Men who develop breast cancer are frequently BRCA1 mutation carriers. Moreover, cancers associated with BRCA1 mutations are frequently triple-negative (negative for estrogen receptor, progesterone receptor and HER2 amplification), which have a very poor prognosis. Experimental data have shown that BRCA1 has a dual role in the mammary epithelium in both DNA damage repair and in epithelial lineage commitment that contribute to the high frequency of triple-negative breast cancer. Brca1 deficiency in experimental models is also associated with increased phosphorylation of the IGF-1 receptor which in turns, leads to the MAPK signaling activation and results in increased proliferation. We evaluated a novel IGF-1 inhibitor in the Brca1 loxP/loxP C57bl/6 mouse that develops an early mammary gland hyper-proliferative phenotype characterized by aberrant ductal morphogenesis and DCIS around 9 months of age. Centrosome aberrations, defined cells with more than 2 centrosomes, were significantly increased the mammary epithelium of these mice compared to C57bl/6 control mice (19.8%+/-0.8%, n=13 vs 8.4%+/-0.9%, n=6, p<0.001). We also identified a novel population of cells expressing both a luminal and myoepithelial lineage markers present in the Brca1 loxP/loxP C57bl/6 mice that were rare in control mice (22.5%+/-0.9%, n=5 vs 3.9%+/-0.4%, n=7, p<0.001). Four month old Brca1 loxP/loxP and C57bl/6 mice were treated with either pasireotide (aka SOM230), a multiligand somatostatin analog that directly blocks IGF-1 action in the mammary gland or PQ401, a IGF-IR small molecule inhibitor, administered for 7 days by subcutaneous Alzet pumps Pasireotide and PQ401 inhibited IGF-1 mediated phosphorylation of target proteins, IGF-IR, ERK and AKT in the Brca1 mammary glands. Both treatments resulted in rapid reversion of the Brca1 mutant gross morphological phenotype.!

Accumulating evidence indicates that the malignant behavior of tumors depends not only on tumor cells themselves, but also on the stromal cells that comprise the malignant tumor mass. Experimental findings suggest that stromal cells such as cancer-associated fibroblast (CAF) may play important roles in promoting tumor growth and metastasis as well as regulating the efficacy of certain chemotherapeutic drugs. However, developing novel clinical strategies that selectively and simultaneously impacts tumor and stromal cells remains challenging. Alterations in the integrity and molecular composition of the extracellular matrix (ECM) are hallmarks of tumor progression. Our previous studies have shown that structural remodeling of the ECM can result in localized triggering of what we have termed "biomechanical ECM switches" or changes in the three-dimensional structure of pre-existing ECM molecules. A humanized antibody (TRC093/D93) specifically directed to the HU177 cryptic collagen epitope that is selectively exposed following triggering of a biomechanical ECM switch has been developed, and a human phase-I clinical trial was recently completed with encouraging results. Here we provide evidence that the HU177 biomechanical ECM switch is triggered within human ovarian tumors resulting in the exposure of the HU177 cryptic collagen epitope. The relative exposure of the HU177 cryptic site was significantly (P<0.05) enhanced in biopsies of malignant ovarian tumors as compared to benign ovarian lesions. Selective targeting of the HU177 cryptic collagen epitope by Mab D93 significantly (P<0.05) inhibited SKOV-3 tumor growth by approximately 70% as compared to controls. Tumors from these mice exhibited reduced angiogenesis, elevated levels of apoptosis and a significant reduction in infiltration of alpha smooth muscle cell actin (alphaSMC-actin) positive stromal fibroblasts. Importantly, while Mab D93 inhibited SKOV-3 tumor cell adhesion to denatured collagen type-I and enhanced the expression of the cyclin !

Temporal lobe epilepsy (TLE) is associated with some of the same neuropathological features as those reported for early stages of typical Alzheimer's disease (AD). The APOE epsilon4 allele is associated with a gene-dose-dependent increase in AD risk and in the severity of amyloid-beta (Abeta) pathology. In a study published in the current BMC Medicine, Sue Griffin and colleagues studied markers of brain resilience in the amputated temporal lobes of TLE patients. They discovered compelling evidence that the APOE epsilon3 isoform in TLE patients is apparently more neuroprotective from Abeta toxicity than is the APOE epsilon4 isoform, as shown by the reduced levels of neuronal damage, glial activation, and expression of IL-1alpha in the APOE epsilon3/epsilon3 brains. This result points to a new property of APOE isoforms: not only are APOE epsilon4 alleles associated with increased brain amyloid plaque burden, but these alleles are also apparently inferior to APOE epsilon3 alleles in conveying resistance to Abeta neurotoxicity. This 'double whammy' result opens up a new direction for studies aimed at elucidating the relevant neurobiological activities of APOE isoforms in the pathogenesis of AD.