2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.

G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection.

There is currently no efficient method available for the delivery of full length functional proteins into the cytoplasm of retinal cells in vivo. Historically, the most successful approach for the treatment of retinal diseases has been intravitreal injection of antibodies or recombinant proteins, but this approach is not yet utilized for the delivery of proteins that require intracellular access for a therapeutic effect. Here we describe a platform for the delivery of functional proteins into ganglion cells, photoreceptors and retinal pigment epithelium via intravitreal injection. A nucleolin binding aptamer, AS1411, was biotinylated and complexed with traptavidin and utilized as a platform for the delivery of GFP or X-linked inhibitor of apoptosis (XIAP) proteins by intravitreal injection in BALB/c mice. Retinal sections were analyzed for uptake of proteins in the retina. Apoptosis was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Retinas were harvested for analysis of TUNEL and caspase 3/7 activity. Intravitreal injection of AS1411-directed GFP or XIAP complexes enabled delivery of these proteins into ganglion cells, photoreceptors and retinal pigment epithelium in vivo. AS1411-XIAP complexes conferred significant protection to cells in the outer and inner nuclear layers following NMDA induced apoptosis. A concomitant decrease in activity of Caspase 3/7 was observed in eyes injected with the AS1411-XIAP complex. In conclusion, AS1411 can be used as a platform for the delivery of therapeutic proteins into retinal cells. This approach can potentially be utilized to introduce a large variety of therapeutically relevant proteins that are previously well characterized to maintain the structural integrity and function of retina, thus, preventing vision loss due to ocular trauma or inherited retinal degeneration.

Reducible PEG-POD/DNA Nanoparticles for Gene Transfer In Vitro and In Vivo: Application in a Mouse Model of Age-Related Macular Degeneration.

Non-viral gene delivery systems are being developed to address limitations of viral gene delivery. Many of these non-viral systems are modeled on the properties of viruses including cell surface binding, endocytosis, endosomal escape, and nuclear targeting. Most non-viral gene transfer systems exhibit little correlation between in vitro and in vivo efficiency, hampering a systematic approach to their development. Previously, we have described a 3.5 kDa peptide (peptide for ocular delivery [POD]) that targets cell surface sialic acid. When functionalized with polyethylene glycol (PEG) via a sulfhydryl group on the N-terminal cysteine of POD, PEG-POD could compact plasmid DNA, forming 120- to 180-nm homogeneous nanoparticles. PEG-POD enabled modest gene transfer and rescue of retinal degeneration in vivo. Systematic investigation of different stages of gene transfer by PEG-POD nanoparticles was hampered by their inability to deliver genes in vitro. Herein, we describe functionalization of POD with PEG using a reducible orthopyridyl disulfide bond. These reducible nanoparticles enabled gene transfer in vitro while retaining their in vivo gene transfer properties. These reducible PEG-POD nanoparticles were utilized to deliver human FLT1 to the retina in vivo, achieving a 50% reduction in choroidal neovascularization in a murine model of age-related macular degeneration.

Differential effects of the estrogen receptor agonist estradiol on toxicity induced by enzymatically-derived or autoxidation-derived oxysterols in human ARPE-19 cells.

PURPOSE/AIM OF THE STUDY: Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER. MATERIALS AND METHODS: ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels. RESULTS: Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC. CONCLUSIONS: Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.

Deferiprone reduces amyloid-ß and tau phosphorylation levels but not reactive oxygen species generation in hippocampus of rabbits fed a cholesterol-enriched diet.

Accumulation of amyloid-ß (Aß) peptide and the hyperphosphorylation of tau protein are major hallmarks of Alzheimer's disease (AD). The causes of AD are not well known but a number of environmental and dietary factors are suggested to increase the risk of developing AD. Additionally, altered metabolism of iron may have a role in the pathogenesis of AD. We have previously demonstrated that cholesterol-enriched diet causes AD-like pathology with iron deposition in rabbit brain. However, the extent to which chelation of iron protects against this pathology has not been determined. In this study, we administered the iron chelator deferiprone in drinking water to rabbits fed with a 2% cholesterol diet for 12 weeks. We found that deferiprone (both at 10 and 50 mg/kg/day) significantly decreased levels of Aß40 and Aß42 as well as BACE1, the enzyme that initiates cleavage of amyloid-ß protein precursor to yield Aß. Deferiprone also reduced the cholesterol diet-induced increase in phosphorylation of tau but failed to reduce reactive oxygen species generation. While deferiprone treatment was not associated with any change in brain iron levels, it was associated with a significant reduction in plasma iron and cholesterol levels. These results demonstrate that deferiprone confers important protection against hypercholesterolemia-induced AD pathology but the mechanism(s) may involve reduction in plasma iron and cholesterol levels rather than chelation of brain iron. We propose that adding an antioxidant therapy to deferiprone may be necessary to fully protect against cholesterol-enriched diet-induced AD-like pathology.

Endoplasmic reticulum stress-induced CHOP activation mediates the down-regulation of leptin in human neuroblastoma SH-SY5Y cells treated with the oxysterol 27-hydroxycholesterol.

Epidemiological studies have suggested an inverse relationship between the adipocytokine leptin and the onset of Alzheimer's disease (AD), and leptin supplementation decreases amyloid-ß (Aß) production and tau phosphorylation (p-tau), two major biochemical events that play a key role in the pathogenesis of AD. We have previously shown that the cholesterol oxidized product 27-hydroxycholesterol (27-OHC) inhibits leptin expression, an effect that correlated with increased levels of Aß and p-tau. We have also shown that 27-OHC induces endoplasmic reticulum (ER) stress, a cellular response that is implicated in AD and confers leptin resistance. However the extent to which ER stress is involved in 27-OHC-induced attenuation in leptin expression has not been determined. In this study we determined the involvement of ER stress in the 27-OHC-induced attenuation of leptin expression in SH-SY5Y human neuroblastoma cells. We demonstrate that 27-OHC-induced ER stress attenuates leptin expression by activating C/EBP Homologous Protein (CHOP) which negatively regulates C/EBPα, a transcription factor required for leptin expression. The molecular chaperone 4-phenylbutyric acid (4-PBA) precludes 27-OHC-evoked ER stress and down-regulation of leptin. Furthermore, we demonstrate that the activation of the transcription factor CHOP in response to ER stress is pivotal in the attenuation of leptin expression as knocking-down CHOP alleviates the attenuation in leptin expression. Our study implicates ER stress as the mechanistic link in the 27-OHC-induced negative regulation of leptin, a hormone that has potential therapeutic effects in AD by reducing Aß and phosphorylated tau accumulation.

Cholesterol-enriched diet causes age-related macular degeneration-like pathology in rabbit retina.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological hallmarks including ß-amyloid (Aß) accumulation, oxidative stress, and apoptotic cell death. The causes of AD and AMD are likely multi-factorial with several factors such as diet, environment, and genetic susceptibility participating in the pathogenesis of these diseases. Epidemiological studies correlated high plasma cholesterol levels with high incidence of AD, and feeding rabbits with a diet rich in cholesterol has been shown to induce AD-like pathology in rabbit brain. High intake of cholesterol and saturated fat were also long been suspected to increase the risk for AMD. However, the extent to which cholesterol-enriched diet may also cause AMD-like features in rabbit retinas is not well known. METHODS: Male New Zealand white rabbits were fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. At necropsy, animals were perfused with Dulbecco's phosphate-buffered saline and the eyes were promptly removed. One eye of each animal was used for immunohistochemistry and retina dissected from the other eye was used for Western blot, ELISA assays, spectrophotometry and mass spectrometry analyses. RESULTS: Increased levels of Aß, decreased levels of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic Bax and gadd153 proteins, emergence of TUNEL-positive cells, and increased generation of reactive oxygen species were found in retinas from cholesterol-fed compared to normal chow-fed rabbits. Additionally, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. As several lines of evidence suggest that oxidized cholesterol metabolites (oxysterols) may be the link by which cholesterol contributes to the pathogenesis of AMD, we determined levels of oxysterols and found a dramatic increase in levels of oxysterols in retinas from cholesterol-fed rabbits. CONCLUSIONS: Our results suggest that cholesterol-enriched diets cause retinal degeneration that is relevant to AMD. Furthermore, our data suggests high cholesterol levels and subsequent increase in the cholesterol metabolites as potential culprits to AMD.

Molecular interplay between leptin, insulin-like growth factor-1, and ß-amyloid in organotypic slices from rabbit hippocampus.

BACKGROUND: Evidence shows that the insulin-like growth factor-1 (IGF-1) and leptin reduce ß-amyloid (Aß) production and tau phosphorylation, two major hallmarks of Alzheimer's disease (AD). IGF-1 expression involves the JAK/STAT pathway and the expression of leptin is regulated by the mammalian target of rapamycin complex 1 (mTORC1). We have previously shown that Aß reduces leptin by inhibiting the mTORC1 pathway and Aß was also suggested to inhibit the JAK/STAT pathway, potentially attenuating IGF-1 expression. As IGF-1 can activate mTORC1 and leptin can modulate JAK/STAT pathway, we determined the extent to which IGF-1 and leptin can upregulate the expression of one another and protect against Aß-induced downregulation. RESULTS: We demonstrate that incubation of organotypic slices from adult rabbit hippocampus with Aß42 downregulates IGF-1 expression by inhibiting JAK2/STAT5 pathway. Leptin treatment reverses these Aß42 effects on IGF-1 and treatment with the STAT5 inhibitor completely abrogated the leptin-induced increase in IGF-1. Furthermore, EMSA and ChIP analyses revealed that leptin increases the STAT5 binding to the IGF-1 promoter. We also show that IGF-1 increases the expression of leptin and reverses the Aß42-induced attenuation in leptin expression via the activation of mTORC1 signaling as the mTORC1 inhibitor rapamycin completely precluded the IGF-1-induced increase in leptin expression. CONCLUSION: Our results demonstrate for the first time that Aß42 downregulates IGF-1 expression and that leptin and IGF-1 rescue one another from downregulation by Aß42. Our study provides a valuable insight into the leptin/IGF-1/Aß interplay that may be relevant to the pathophysiology of AD.

The oxysterol 27-hydroxycholesterol increases ß-amyloid and oxidative stress in retinal pigment epithelial cells.

BACKGROUND: Alzheimer's disease (AD) and age-related macular degeneration (AMD) share several pathological features including ß-amyloid (Aß) peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD. METHODS: ARPE-19 cells were treated with 0, 10 or 25 µM 27-OHC for 24 hours. Levels of Aß peptide, mitochondrial and endoplasmic reticulum (ER) stress markers, Ca2+ homeostasis, glutathione depletion, reactive oxygen species (ROS) generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays. RESULTS: 27-OHC dose-dependently increased Aß peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP), reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, increased levels of the nuclear factor κB (NFκB) and heme-oxygenase 1 (HO-1), two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death. CONCLUSIONS: The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aß accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for both AMD and AD.

ß-Amyloid regulates leptin expression and tau phosphorylation through the mTORC1 signaling pathway.

High levels of the adipocytokine leptin are associated with reduced risk of Alzheimer's disease. Leptin treatment also reduces ß-amyloid (Aß) levels in in vivo and in vitro models of Alzheimer's disease. Aß and leptin interact with the Akt/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Akt/mTORC1 activation reduces tau phosphorylation through the inhibition of the downstream enzyme GSK-3ß. mTORC1 also regulates translation of many proteins including leptin. While Aß has been shown to inactivate Akt, inhibit mTORC1, and facilitate the phosphorylation of tau, leptin activates both Akt and mTORC1 and reduces tau phosphorylation. However, the extent to which Aß may modulate leptin expression and increase tau phosphorylation involving Akt/mTORC1 has not been determined. In this study, we show that incubation of organotypic slices from rabbit hippocampus with Aß down-regulates leptin expression, inhibits Akt, activates GSK-3ß, increases tau phosphorylation, and inactivates mTORC1. Leptin treatment reverses Aß effects by alleviating Akt inhibition, preventing GSK-3ß activation, reducing tau phosphorylation, and activating mTORC1. On the other hand, Rapamycin, an allosteric inhibitor of mTORC1, down-regulates leptin expression, increases tau phosphorylation, and does not affect Akt and GSK-3ß. Our results demonstrate for the first time that Aß regulates leptin expression and tau phosphorylation through mTORC1.

Caffeine protects against oxidative stress and Alzheimer's disease-like pathology in rabbit hippocampus induced by cholesterol-enriched diet.

Cholesterol has been linked to the pathogenesis of sporadic Alzheimer's disease (AD) as a risk factor increasing beta-amyloid (Abeta) and oxidative stress levels. Caffeine has antioxidant properties and has been demonstrated to reduce Abeta levels in transgenic mouse models of familial AD. However, the effects of caffeine on cholesterol-induced sporadic AD pathology have not been determined. In this study, we determined the effects of caffeine on Abeta levels, tau phosphorylation, oxidative stress generation, and caffeine-target receptors in rabbits fed a 2% cholesterol-enriched diet, a model system for sporadic AD. Our results showed that the cholesterol-enriched diet increased levels of Abeta, tau phosphorylation, and oxidative stress measured as increased levels of reactive oxygen species and isoprostanes, glutathione depletion, and increased levels of endoplasmic reticulum stress marker proteins. Additionally, the cholesterol-enriched diet reduced the levels of adenosine A(1) receptors (A(1)R) but not ryanodine or adenosine A(2A) receptors. Caffeine, administered at 0.5 and 30mg/day in the drinking water, reduced the cholesterol-induced increase in Abeta, phosphorylated tau, and oxidative stress levels and reversed the cholesterol-induced decrease in A(1)R levels. Our results suggest that even very low doses of caffeine might protect against sporadic AD-like pathology.

Leptin reduces the accumulation of Abeta and phosphorylated tau induced by 27-hydroxycholesterol in rabbit organotypic slices.

Accumulation of amyloid-beta (Abeta) peptide and deposition of hyperphosphorylated tau protein are two major pathological hallmarks of Alzheimer's disease (AD). We have shown that cholesterol-enriched diets and its metabolite 27-hydroxycholesterol (27-OHC) increase Abeta and phosphorylated tau levels. However, the mechanisms by which cholesterol and 27-OHC regulate Abeta production and tau phosphorylation remain unclear. Leptin, an adipocytokine involved in cell survival and in learning, has been demonstrated to regulate Abeta production and tau hyperphosphorylation in transgenic mice for AD. However, the involvement of leptin signaling in cholesterol and cholesterol metabolites-induced Abeta accumulation and tau hyperphosphorylation are yet to be examined. In this study, we determined the effect of high cholesterol diet and 27-OHC on leptin expression levels and the extent to which leptin treatment affects 27-OHC-induced AD-like pathology. Our results show that feeding rabbits a 2% cholesterol-enriched diet for 12 weeks reduces the levels of leptin by approximately 80% and incubating organotypic slices from adult rabbit hippocampus with 27-OHC reduced leptin levels by approximately 30%. 27-OHC induces a 1.5-fold increase in Abeta (40) and a 3-fold increase in Abeta (42) and in phosphorylated tau. Treatment with leptin reversed the 27-OHC-induced increase in Abeta and phosphorylated tau by decreasing the levels of BACE-1 and GSK-3beta respectively. Our results suggest that cholesterol-enriched diets and cholesterol metabolites induce AD-like pathology by altering leptin signaling. We propose that leptin administration may prevent the progression of sporadic forms of AD that are related to increased cholesterol and oxidized cholesterol metabolite levels.