Faculty Bibliography

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

Aims Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to progressive accumulation of neurotoxic amyloid beta (Abeta) oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Method We have used a combination of stereotaxic injection into the hippocampal region of C57BL/6 wild-type mice with biochemical and mass spectrometric analyses of CSF to evaluate the brain clearance and catabolism of well-defined monomeric and low molecular mass Abeta oligomeric assemblies. Results Abeta physiologic removal from the brain is extremely fast, involves local proteolytic degradation with generation of heterogeneous C-terminally cleaved proteolytic products, and is negatively influenced by oligomerization. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. Clearance from brain interstitial fluid follows local and systemic paths; in addition to the BBB, local enzymatic degradation and transport through the choroid plexus into the CSF play significant roles. Conclusion Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies

Aims Biochemical and proteomic analysis of brain deposits and biological fluids reveal a high degree of Abeta heterogeneity that goes far beyond the classical Abeta40/Abeta42 dichotomy, displaying numerous post-translational modifications and multiple truncations at both N- and C-terminal ends of the molecule likely reflecting local action of resident enzymes. In spite of innumerable studies focusing in Abeta, the relevance of N- and C-terminal truncated species in the mechanism of AD pathogenesis remains largely understudied. Method Abeta species in brain tissue extracts were identified via immunoprecipitation/mass spectrometry. Synthetic homologues of intact and truncated peptides were compared in their solubility properties, self-oligomerization propensity, and brain clearance characteristics. Novel antibodies recognizing specific N- and C-terminal truncations were employed to immunolabel amyloid deposits in AD brains and transgenic models. Intracerebral injections of monomeric and oligomeric radiolabeled homologues were used to assess their brain clearance characteristics. Results N- and C-terminal truncated fragments in brain homogenates exhibit differential fractionation characteristics and topographic localization. Water-soluble brain extracts were enriched in C-terminal fragments -resembling the CSF Abeta peptidome- whereas N-terminal truncations required formic acid for solubilization. Synthetic homologues confirmed the differences in solubility and revealed contrasting oligomerization/ fibrillization characteristics. Notably, oligomerization largely increased brain retention, a characteristic mostly evident in fragments truncated at Phe4, topographically abundant in the plaque cores. Conclusion Abeta degradation at the C-terminal-end generates fragments likely associated to catabolic/clearance mechanisms while truncations at the N-terminus favor oligomerization and brain retention, with the potential to exacerbate the process of amyloidogenesis

Mitochondrial dysfunction has been recognized as an early event in Alzheimer's disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington's disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Abeta) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Abeta, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Abeta in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Abeta injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Abeta-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

Familial Danish Dementia (FDD), an early-onset non-amyloid-beta (Abeta) cerebral amyloidosis, is neuropathologically characterized by widespread cerebral amyloid angiopathy, parenchymal amyloid and preamyloid deposits, as well as neurofibrillary degeneration indistinguishable to that seen in Alzheimer's disease (AD). The main amyloid subunit composing FDD lesions, a 34-amino acid de-novo generated peptide ADan, is the direct result of a genetic defect at the 3'-end of the BRI2 gene and the physiologic action of furin-like proteolytic processing at the C-terminal region of the ADan precursor protein. We aimed to study the impact of the FDD mutation, the additional formation of the pyroglutamate (pE) posttranslational modification as well as the relevance of C-terminal truncations -all major components of the heterogeneous FDD deposits- on the structural and neurotoxic properties of the molecule. Our data indicates that whereas the mutation generated a beta-sheet-rich hydrophobic ADan subunit of high oligomerization/fibrillization propensity and the pE modification further enhanced these properties, C-terminal truncations had the opposite effect mostly abolishing these features. The potentiation of pro-amyloidogenic properties correlated with the initiation of neuronal cell death mechanisms involving oxidative stress, perturbation of mitochondrial membrane potential, release of mitochondrial cytochrome c, and downstream activation of caspase-mediated apoptotic pathways. The amyloid-induced toxicity was inhibited by targeting specific components of these detrimental cellular pathways, using reactive oxygen scavengers and monoclonal antibodies recognizing the pathological amyloid subunit. Taken together, the data indicate that the FDD mutation and the pE posttranslational modification are both primary elements driving intact ADan into an amyloidogenic/neurotoxic pathway while truncations at the C-terminus eliminate the pro-amyloidogenic characteristics of the molecule, likely reflecting effect of physiologic clearance mechanisms.

Amyloid beta (Abeta) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Abeta oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Abeta1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Abeta physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Abeta efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

Background: Neurodegeneration and memory loss in Alzheimer's disease (AD) have been associated in many reports with mitochondrial dysfunction. Molecular pathways triggered by mitochondrial deregulation, with associated production of reactive oxygen species and release of pro-apoptotic factors, are thought to be early events in the pathogenesis of the disease. Carbonic anhydrase inhibitors such as methazolamide (MTZ) are FDA approved for glaucoma as well as other indications, and have been considered as potential therapeutic strategies in models of Huntington's disease, stroke, muscular dystrophy and diabetes. Methods: We analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer's amyloid in vitro and by intra-hippocampal injection resulting in interstitial and cellular accumulation of Abeta in the mouse brain. Results:MTZ prevented CytC release and activation of caspase 9 and caspase 3 induced by Abeta in neuronal and glial cells through the inhibition of mitochondrial hydrogen peroxide production, and inhibited DNA fragmentation indicative of apoptosis. MTZ administered IP before intra-hippocampal Abeta injection was effective at reducing caspase 3 activation and neurodegeneration in the mouse brain. Conclusions: Our results demonstrate the efficacy of MTZ in in vitro and in vivo models of amyloid-mediated toxicity, delineating the molecular mechanism of action of the compound and providing the first evidence in support of the possibility of a new therapeutic approach for AD

Matrix metalloproteases MMP-2 and MMP-9 have been implicated in the physiologic catabolism of Alzheimer amyloid-beta (Abeta). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathologic processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Abeta40 and Abeta42 and the resulting proteolytic fragments assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Abeta fragments truncated only at the C-terminus, ending at positions 34, 30 and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Abeta42 by rhMMP-2 while the enzyme cleaved >80% of Abeta40 during the first hour of incubation. rhMMP-9 was significantly less effective, particularly in degrading Abeta1-42, although the targeted peptide bonds were identical. Using Abeta1-34 and Abeta1-30, we demonstrated that these peptides are also substrates for both MMPs, cleaving Abeta1-34 to produce Abeta1-30 first and Abeta1-16 subsequently. Consistent with the kinetics observed with full-length Abeta, rhMMP-9 degraded only a minute fraction of Abeta1-34 and was even less effective in producing Abeta1-16. Further degradation of Abeta1-16 by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Abeta fragments were highly soluble, did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Abeta species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process.

Objective: A heterogeneous group of amyloid beta (Abeta) species constitute the parenchymal and cerebrovascular amyloid deposits in Alzheimer's disease (AD). Besides the classic full-length peptides, biochemical and proteomic analysis of AD deposits revealed high degree of Abeta heterogeneity at both N- and C-terminal ends likely resulting from the local action of multiple proteolytic enzymes. Interestingly, many of these fragments are also normal components of cerebrospinal fluid, suggesting their active participation in clearance mechanisms. Increasing evidence indicates that deficient brain clearance largely contributes to Abeta accumulation; thus, we compared the biophysical properties and lesion distribution of various N- and C-terminally degraded Abeta fragments to better understand their biological importance. Methods: Synthetic homologues of in vivo identified truncated peptides were used to compare solubility properties, formation of beta-sheet-rich structures, binding to thioflavin T, self-oligomerization and formation of amyloid-like fibrils. Novel antibodies recognizing specific N- and C-terminal truncations were generated and employed to immunolabel amyloid deposits and conduct biochemical analysis in transgenic models and AD brains. Results: N- and C-terminally truncated peptides exhibited completely different biophysical properties and brain tissue distribution. C-terminally degraded Abeta fragments were extremely soluble, did not convert to beta-sheet-rich structures, failed to aggregate or form fibrils and did not co-localize with plaque deposits. Contrastingly, those degraded at the N-terminus were poorly soluble, with high tendency to aggregate and fibrillize and specifically co-localize with Congo-red-positive plaque cores. Conclusions: Degradation at the C-terminal-end of Abeta generates fragments likely associated to catabolic/clearance mechanisms while truncations at the N-terminus favor the process of amyloidogenesis

Objective: Vascular deposition of amyloid, a universal feature in Alzheimer's disease (AD), severely compromises the integrity of the neurovascular unit, a dynamic entity encompassing functional interactions among cells of the microvasculature, neurons, and astrocytic/glial populations. The complexity of the cellular mechanisms elicited by amyloid in astrocytic/glial cells and their relationship to the induction of pro-inflammatory conditions capable of affecting microvessel function/permeability remain to be fully elucidated. In this work we aimed to provide insight into the molecular pathways affected and identify potential new targets for drug discovery. Methods: Astrocytic/glioma cells were challenged with wild-type Abeta and the E22Q vasculotropic variant associated with cerebrovascular deposition and hemorrhagic clinical phenotypes. A combination of FACs-analyzed bead arrays, ELISA, zymography, and confocal studies were employed to evaluate production of pro-inflammatory cytokines, activation of MMPs, and ROS generation whereas the vitamin-E analog Trolox was tested for prevention/amelioration of these detrimental cellular pathways. Results: Oligomeric-Abeta triggered elevated production of the pro-inflammatory mediators IL-6, IL-8, and IFN-gamma, enhanced activation of MMP-2, exacerbated ROS production, and cell death. In all cases, challenge with E22Q translated into a more pronounced response, in agreement with the high oligomerization tendency of the variant and the aggressiveness and early onset of the clinical phenotype. Trolox not only inhibited ROS production and MMP-2 activation, but also preserved cellular integrity and viability, highlighting the primary role of ROS in the initiation of amyloid-induced cell death pathways. Conclusions: Our data emphasizes the detrimental role of astrocyte/glia-initiated Abeta-mediated pro-inflammatory pathways for the integrity of the neurovascular unit