Kimberly S. Fuson

Functional gamma‐secretase inhibitors reduce beta‐amyloid peptide levels in brain

Converging lines of evidence implicate the beta‐amyloid peptide (Aβ) as causative in Alzheimers disease. We describe a novel class of compounds that reduce Aβ production by functionally inhibiting γ‐secretase, the activity responsible for the carboxy‐terminal cleavage required for Aβ production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Aβ production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, to mice transgenic for human APPV717F reduces brain levels of Aβ in a dose‐dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain Aβin vivo. Development of such novel functional γ‐secretase inhibitors will enable a clinical examination of the Aβ hypothesis that Aβ peptide drives the neuropathology observed in Alzheimers disease.

Neurotoxicity of Human Amylin in Rat Primary Hippocampal Cultures: Similarity to Alzheimer's Disease Amyloid-β Neurotoxicity

Amylin, a 37‐amino‐acid amyloidogenic peptide, bears biophysical similarities to the amyloid‐β peptide (Aβ) deposited in Alzheimers disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with Aβ(1–40). Treatment with human amylin (1–37) resulted in prominent toxicity as assessed by phasecontrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin‐induced neurotoxicity was morphologically similar to that induced by Aβ(1–40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full‐length human amylin was toxic; various amylin peptide fragments including amino acid residues 20–29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and Aβ can adopt a similar neurotoxic conformation in vitro. Similar conformation‐dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of Aβ in Alzheimers disease.

Clusterin (Apo J) Protects Against In Vitro Amyloid-β(1–40) Neurotoxicity

Abstract: Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimers disease (AD), where it is found associated with amyloid‐β (Aβ) plaques. Clusterin also coprecipitates with Aβ from CSF, suggesting a physiological interaction with Aβ. Given this interaction with Aβ, the goal of this study was to determine whether clusterin could modulate Aβ neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC‐9, a full‐length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose‐dependent neuroprotection against Aβ(1–40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of Aβ(1–40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid‐β plaque pathology.

Amyloid β-Mediated Oxidative and Metabolic Stress in Rat Cortical Neurons: No Direct Evidence for a Role for H2O2 Generation

Abstract: H2O2 and free radical‐mediated oxidative stresses have been implicated in mediating amyloid β(1–40) [Aβ(1–40)] neurotoxicity to cultured neurons. In this study, we confirm that addition of the H2O2‐scavenging enzyme catalase protects neurons in culture against Aβ‐mediated toxicity; however, it does so by a mechanism that does not involve its ability to scavenge H2O2. Aβ‐mediated elevation in intracellular H2O2 production is suppressed by addition of a potent H2O2 scavenger without any significant neuroprotection. Three intracellular biochemical markers of H2O2‐mediated oxidative stress were unchanged by Aβ treatment: (a) glyceraldehyde‐3‐phosphate dehydrogenase activity, (b) hexose monophosphate shunt activity, and (c) glucose oxidation via the tricarboxylic acid cycle. Ionspray mass spectra of Aβ in the incubation medium indicated that Aβ itself is an unlikely source of reactive oxygen species. In this study we demonstrate that intracellular ATP concentration is compromised during the first 24‐h exposure of neurons to Aβ. Our results challenge a pivotal role for H2O2 generation in mediating Aβ toxicity, and we suggest that impairment of energy homeostasis may be a more significant early factor in the neurodegenerative process.

Characterization of 8‐Epiprostaglandin F2α as aMarker of Amyloid β‐Peptide‐Induced Oxidative Damage

Abstract : The amyloid β‐peptide (Aβ) is a major component of the neuritic plaques that are a defining histological characteristic of Alzheimers disease. Aβ can be directly toxic and pro‐inflammatory to cells in vitro. Numerous reports have shown that oxidative damage and reactive oxygen species play a role in Aβ‐mediated neurotoxicity. 8‐Epiprostaglandin F2α (8‐isoprostane) is a well characterized product of lipid peroxidation that is formed nonenzymatically in cell membranes following an oxidative insult. We report a time‐ and concentration‐dependent increase in 8‐isoprostane levels in rat hippocampal cultures treated with Aβ(1‐40) or hydrogen peroxide. As evidence that 8‐isoprostane production is part of an Aβ toxic pathway, alkaline‐treated peptide, which shows minimal toxic activity, resulted in greatly attenuated 8‐isoprostane production. Although the increase in 8‐isoprostane levels preceded cell death, exogenously added 8‐isoprostane had no cytotoxic effects. The antioxidants vitamin E and propyl gallate attenuated Aβ‐induced 8‐isoprostane formation yet had no effect on Aβ‐induced lactate dehydrogenase release. Neither vitamin E nor propyl gallate had any effect on Aβs ability to adopt a β‐pleated sheet structure and deposit on cells as determined by thioflavine S fluorescence. We conclude that 8‐isoprostane is an indicator of Aβ‐induced damage but not necessarily a mediator of Aβ‐induced neurotoxicity. Also, 8‐isoprostane could be a useful marker for assessing oxidative damage in the CNS.

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus. SGP-2 protein levels assessed by immunoblots were markedly increased in both brain regions following ischemia. GFAP RNA levels also increased over 5 fold in caudate nucleus and hippocampus following the ischemic insult. Despite significant elevations in GFAP RNA, protein levels of GFAP assessed by immunoblot were only marginally affected. The elevated expression of SGP-2 in rodent brain following this and other experimental lesion paradigms (e.g., excitotoxic lesions, deafferentation) suggest some general involvement of SGP-2 in neurodegeneration and remodelling following neuronal injury.

Fibroblast growth factor-8 protects cultured rat hippocampal neurons from oxidative insult.

Basic fibroblast growth factor (bFGF) has been reported to have neuroprotective properties following excitotoxic, metabolic, and oxidative insults. We report here that another FGF family member, FGF-8 is able to protect rat hippocampal cultures from oxidative stress. The b isoform of FGF-8 protected hippocampal cultures from hydrogen peroxide with an EC50 of approximately 25 ng/ml. In a time course study, using pre-, co-, post-treatment paradigms, we report that bFGF and FGF-8b were neuroprotective when added as a pre-treatment, co-treatment, and even at 2 h post-insult. Using neuronal enriched cultures, we demonstrate that bFGF and FGF-8b neuroprotection partially results from a direct action of the growth factors on neurons. The direct action on neurons may work in concert with normal and FGF-stimulated glial secretion products to give the full FGF protective effect. FGF-8b showed maximal protection at 50 ng/ml, whereas bFGF showed maximal protection at 10 ng/ml. Despite requiring higher concentrations to elicit protection, FGF-8b is able to attain levels of protection equivalent to that of bFGF (attenuation of 75-80% of hydrogen peroxide induced death). We also report that bFGF and FGF-8b are able to protect the human neuroblastoma cell line, SK-N-MC, from peroxide-induced LDH release by 50%. From these studies, we conclude that FGF-8b is another member of the FGF family which may show in vivo efficacy for the treatment of oxidative insults, such as stroke.

Characterization of LY231617 Protection Against Hydrogen Peroxide Toxicity

Abstract : The compound LY231617 {2,6‐bis(1,1‐dimethylethyl)‐4‐[[(1‐ethyl)amino]methyl]phenol hydrochloride} has been reported to afford significant neuroprotection against hydrogen peroxide (H2O2)‐induced toxicity in vitro and global ischemia in vivo. We now report on further mechanistic studies of H2O2 toxicity and protection by LY231617. Brief exposure to H2O2 (15 min) elicited an oxidative insult comparable with that generated by overnight treatment. H2O2‐mediated cellular degeneration was characterized using lactate dehydrogenase (LDH) release, changes in total glutathione, and a new marker of oxidative stress, 8‐epiprostaglandin F2α (8‐isoprostane). LY231617 attenuated H2O2‐mediated degeneration under a variety of exposure conditions, including a more clinically relevant posttreatment paradigm. Levels of 8‐isoprostane paralleled LDH release under various treatment paradigms of 100 μM H2O2± 5 μM drug. In contrast, despite affording significant protection, LY231617 had modest to no effects on cellular levels of glutathione. Taken together, these results are consistent with a membrane site of action for LY231617 and suggest that the compound affords cytoprotection via its antioxidant properties.

Stereoselective neuroprotection by novel 2,3-benzodiazepine non-competitive AMPA antagonists against non-NMDA receptor-mediated excitotoxicity in primary rat hippocampal cultures

Glutamate excitotoxicity has been implicated in a variety of acute and chronic neurodegenerative diseases but early phase clinical trials with competitive antagonists at both N-methyl-D-aspartate (NMDA)-receptors and alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors have been disappointing. A family of atypical 2,3 benzodiazepines, exemplified by GYKI 52466, have been described recently which function as non-competitive AMPA-receptor antagonists. We have investigated the neuroprotective efficacy of LY303070 and LY300164, two analogs of GYKI-52466, in an embryonic rat hippocampal culture model of non-NMDA receptor-mediated excitotoxicity using kainic acid (KA) as an agonist at the AMPA/KA receptor. Overnight treatment with 500 microM KA resulted in prominent neuronal excitotoxicity as assessed by lactate dehydrogenase efflux. LY300164 and LY303070 attenuated KA-excitotoxicity in a dose-dependent manner with IC50s of 4 and 2 microM, respectively. In contrast, their stereoisomers, LY300165 and LY303071 showed no neuroprotection at concentrations up to 25 microM. In addition, AMPA-mediated excitotoxicity in cyclothiazide pre-treated cultures was also completely blocked by LY303070. Finally, neuroprotection by this class of 2,3 benzodiazepines was not influenced by antagonism of the classical benzodiazepine receptor. LY303070 and LY300164 represent novel non-competitive AMPA-receptor antagonists which may offer unique advantages in the clinic over competitive AMPA-receptor antagonists.

Induction of sulfated glycoprotein-2 (clusterin) and glial fibrillary acidic protein (GFAP) RNA expression following transient global ischemia is differentially attenuated by LY231617.

Sulfated glycoprotein-2 (SGP-2) is a secreted glycoprotein that along with GFAP has emerged as a prominent molecular marker of neurodegeneration. In the present study, we have evaluated further the relationship between SGP-2, GFAP and neurodegeneration, by examining the effects of LY231617, a potent antioxidant, on expression of SGP-2 and GFAP following four vessel occlusion (4VO). GFAP and SGP-2 RNA levels increased several fold in hippocampus and caudate nucleus in response to 30 min of 4VO. LY231617 treatment markedly attenuated the induction of GFAP RNA in both hippocampus and caudate nucleus, consistent with the significant neuroprotection observed histologically. In contrast, LY231617 treatment blunted SGP-2 RNA expression only in the hippocampus; SGP-2 RNA expression in caudate nucleus was similar to vehicle-treated 4VO, despite the marked attenuation of neuronal damage in both areas by LY231617. These data suggest region-specific differential regulation of SGP-2 and GFAP RNA induction.