Novel brain-targeted nanomicelles for anti-glioma therapy mediated by the ApoE-enriched protein corona in vivo.

BACKGROUND: The interactions between nanoparticles (NPs) and plasma proteins form a protein corona around NPs after entering the biological environment, which provides new biological properties to NPs and mediates their interactions with cells and biological barriers. Given the inevitable interactions, we regard nanoparticle‒protein interactions as a tool for designing protein corona-mediated drug delivery systems. Herein, we demonstrate the successful application of protein corona-mediated brain-targeted nanomicelles in the treatment of glioma, loading them with paclitaxel (PTX), and decorating them with amyloid ß-protein (Aß)-CN peptide (PTX/Aß-CN-PMs). Aß-CN peptide, like the Aß1-42 peptide, specifically binds to the lipid-binding domain of apolipoprotein E (ApoE) in vivo to form the ApoE-enriched protein corona surrounding Aß-CN-PMs (ApoE/PTX/Aß-CN-PMs). The receptor-binding domain of the ApoE then combines with low-density lipoprotein receptor (LDLr) and LDLr-related protein 1 receptor (LRP1r) expressed in the blood-brain barrier and glioma, effectively mediating brain-targeted delivery. METHODS: PTX/Aß-CN-PMs were prepared using a film hydration method with sonication, which was simple and feasible. The specific formation of the ApoE-enriched protein corona around nanoparticles was characterized by Western blotting analysis and LC-MS/MS. The in vitro physicochemical properties and in vivo anti-glioma effects of PTX/Aß-CN-PMs were also well studied. RESULTS: The average size and zeta potential of PTX/Aß-CN-PMs and ApoE/PTX/Aß-CN-PMs were 103.1 nm, 172.3 nm, 7.23 mV, and 0.715 mV, respectively. PTX was efficiently loaded into PTX/Aß-CN-PMs, and the PTX release from rhApoE/PTX/Aß-CN-PMs exhibited a sustained-release pattern in vitro. The formation of the ApoE-enriched protein corona significantly improved the cellular uptake of Aß-CN-PMs on C6 cells and human umbilical vein endothelial cells (HUVECs) and enhanced permeability to the blood-brain tumor barrier in vitro. Meanwhile, PTX/Aß-CN-PMs with ApoE-enriched protein corona had a greater ability to inhibit cell proliferation and induce cell apoptosis than taxol. Importantly, PTX/Aß-CN-PMs exhibited better anti-glioma effects and tissue distribution profile with rapid accumulation in glioma tissues in vivo and prolonged median survival of glioma-bearing mice compared to those associated with PMs without the ApoE protein corona. CONCLUSIONS: The designed PTX/Aß-CN-PMs exhibited significantly enhanced anti-glioma efficacy. Importantly, this study provided a strategy for the rational design of a protein corona-based brain-targeted drug delivery system. More crucially, we utilized the unfavorable side of the protein corona and converted it into an advantage to achieve brain-targeted drug delivery.

Incorporation of apolipoprotein E into HBV-HCV subviral envelope particles to improve the hepatitis vaccine strategy.

Hepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV-HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV-HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV-HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins.

Calcification of the cavernosal bodies may be responsible for development of erectile dysfunction in uremic apolipoprotein E deficient (apoE-/-) mice / Calcificación de cuerpos cavernosos y desarrollo de disfunción eréctil en la falta de apolipoproteína urémica E (ratones ApoE-/-) en un modelo de ratón

INTRODUCTION AND OBJECTIVE: Erectile dysfunction's physiopathology in uremia is complex and multifactorial, involving a combination of classical risk factors and specific uremia-related risk factors such as increased oxidative stress, endothelial dysfunction and inflammation. The aim of the study is to investigate the effect of chronic kidney disease (CKD) on vascular calcification and endothelial function of cavernosal bodies in apolipoprotein E deficient (apoE−/−) mice, a well known model of erectile dysfunction. MATERIALS AND METHODS: Eight-week-old male apoE−/− mice were randomly assigned to the following 3 groups: (I) subtotally nephrectomised (SNX apoE−/−, 12 mice), (II) uninephrectomised (UNX apoE−/−, 11 mice) or (III) sham operated (sham-op apoE−/−, 15 mice). At 16 weeks after surgery, aortas and penile erectile tissues were harvested for histological studies to assess atherosclerosis, vascular calcification, nitrotyrosine staining, total collagen content and macrophage staining. RESULTS: At sacrifice, SNX and UNX mice had significantly higher serum urea, total cholesterol, and triglyceride concentrations than sham-op controls. Atherosclerotic lesions in thoracic aorta were significantly larger in uremic apoE−/− mice than in controls. There were no atheromatous lesions in cavernosal bodies or penile artery observed in any group. However, SNX and UNX animals showed a significant increase in calcification score, collagen content and nitrotyrosine staining in cavernosal bodies when compared with controls. The degree of macrophage infiltration was comparable between the 3 groups. CONCLUSION: In conclusion, even mild renal dysfunction, i.e., after uninephrectomy increases calcification score and aggravates endothelial function of cavernosal bodies in apoE−/− mice and this effect might be linked to increased oxidative stress in penile endothelium

INTRODUCCIÓN Y OBJETIVOS: La fisiopatología de la disfunción eréctil en la uremia es compleja y multifactorial, e incluye una combinación de factores de riesgo clásicos y factores específicos asociados a la uremia, como el aumento del estrés oxidativo, la disfunción endotelial y la inflamación. El objetivo de este trabajo es examinar el efecto de la enfermedad renal crónica (ERC) sobre la calcificación vascular y la función endotelial de los cuerpos cavernosos en caso de deficiencia de apolipoproteína E en ratones (ratones ApoE−/−), un modelo bien conocido de disfunción eréctil. MATERIALES Y MÉTODOS: Los ratones machos de 8 semanas de edad con «ApoE−/−mice» se distribuyen aleatoriamente en 3 grupos: 1) con heminefrectomía (SNX ApoE−/−), 12 ratones; 2) con nefrectomía única (UNX ApoE−/−), 11 ratones, y 3) operación de placebo (sham-op ApoE−/−), 15 ratones. Dieciséis semanas después de la cirugía, se retiraron los tejidos eréctiles de la aorta y el pene para realizar estudios histológicos con el fin de evaluar la aterosclerosis, la calcificación vascular, las sombras de nitrotirosina, el contenido de colágeno total y las sombras de macrófagos. RESULTADOS: Durante el sacrificio, los ratones con SNX y UNX reflejaron valores de urea sérica, colesterol total y concentración de triglicéridos significativamente más elevados, en comparación con los casos controlados con placebo. Las lesiones ateroscleróticas en la aorta torácica fueron mucho mayores en los ratones urémicos «ApoE−/−» en comparación con los controles. No hubo lesiones ateromatosas en los cuerpos cavernosos ni en la arteria del pene en ninguno de los grupos. Sin embargo, los animales con nefrectomía seminal y única mostraron un aumento significativo en la calcificación, presencia de colágeno y manchas de nitrotirosina en cuerpos cavernosos en comparación con los controles. El grado de infiltración de macrófagos fue comparable entre los 3 grupos. CONCLUSIÓN: Se ha concluido que incluso una disfunción renal menor, es decir, tras una nefrectomía única, aumenta la calcificación y exacerba la función endotelial de los cuerpos cavernosos en ratones «ApoE−/−», y este efecto puede estar asociado a un aumento del estrés oxidativo en el endotelio del pene

ABCA1/ApoE/HDL Signaling Pathway Facilitates Myelination and Oligodendrogenesis after Stroke.

ATP-binding cassette transporter A1 (ABCA1) plays an important role in the regulation of apolipoprotein E (ApoE) and the biogenesis of high-density lipoprotein (HDL) cholesterol in the mammalian brain. Cholesterol is a major source for myelination. Here, we investigate whether ABCA1/ApoE/HDL contribute to myelin repair and oligodendrogenesis in the ischemic brain after stroke. Specific brain ABCA1-deficient (ABCA1-B/-B) and ABCA1-floxed (ABCA1fl/fl) control mice were subjected to permanent distal middle-cerebral-artery occlusion (dMCAo) and were intracerebrally administered (1) artificial mouse cerebrospinal fluid (CSF) as vehicle control, (2) human plasma HDL3, and (3) recombined human ApoE2 starting 24 h after dMCAo for 14 days. All stroke mice were sacrificed 21 days after dMCAo. The ABCA1-B/-B-dMCAo mice exhibit significantly reduced myelination and oligodendrogenesis in the ischemic brain as well as decreased functional outcome 21 days after stroke compared with ABCA1fl/fl mice; administration of human ApoE2 or HDL3 in the ischemic brain significantly attenuates the deficits in myelination and oligodendrogenesis in ABCA1-B/-B-dMCAo mice ( p < 0.05, n = 9/group). In vitro, ABCA1-B/-B reduces ApoE expression and decreases primary oligodendrocyte progenitor cell (OPC) migration and oligodendrocyte maturation; HDL3 and ApoE2 treatment significantly reverses ABCA1-B/-B-induced reduction in OPC migration and oligodendrocyte maturation. Our data indicate that the ABCA1/ApoE/HDL signaling pathway contributes to myelination and oligodendrogenesis in the ischemic brain after stroke.

Apolipoprotein E promotes white matter remodeling via the Dab1-dependent pathway after traumatic brain injury.

INTRODUCTION: Axonal injury results in long-term neurological deficits in traumatic brain injury (TBI) patients. Apolipoprotein E (ApoE) has been reported to activate intracellular adaptor protein Disabled-1 (Dab1) phosphorylation via its interaction with ApoE receptors. The Dab1 pathway acts as a regulator of axonal outgrowth and growth cone formation in the brain. AIMS: We hypothesized that ApoE may alleviate axonal injury and regulate axonal regeneration via the Dab1 pathway after TBI. RESULTS: In this study, we established a model of controlled cortical impact (CCI) to mimic TBI in vivo. Using diffusion tensor imaging to detect white matter integrity, we demonstrated that APOE-deficient mice exhibited lower fractional anisotropy (FA) values than APOE+/+ mice at 28 days after injury. The expression levels of axonal regeneration and synapse plasticity biomarkers, including growth-associated protein 43 (GAP43), postsynaptic density protein 95 (PSD-95), and synaptophysin, were also lower in APOE-deficient mice. In contrast, APOE deficiency exerted no effects on the levels of myelin basic protein (MBP) expression, oligodendrocyte number, or oligodendrocyte precursor cell number. Neurological severity score (NSS) and behavioral measurements in the rotarod, Morris water maze, and Y maze tests revealed that APOE deficiency caused worse neurological deficits in CCI mice. Furthermore, Dab1 activation downregulation by the ApoE receptor inhibitor receptor-associated protein (RAP) or Dab1 shRNA lentivirus attenuated the beneficial effects of ApoE on FA values, GAP43, PSD-95, and synaptophysin expression, and neurological function tests. Additionally, the effects of ApoE on axonal regeneration were further validated in vitro. In a mechanical scratch injury model of primary cultured neurons, recombinant ApoE protein treatment enhanced axonal outgrowth and growth cone formation in injured neurons; however, these effects were attenuated by Dab1 shRNA, consistent with the in vivo results. CONCLUSION: Collectively, these data suggest that ApoE promotes axonal regeneration partially through the Dab1 pathway, thereby contributing to functional recovery following TBI.

ApoE mimetic improves pathology and memory in a model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is characterized pathologically by Aß plaques. Current treatments are purely symptomatic despite decades of intensive research interest. Notably, patients with the APOE4 allele are at increased risk for developing AD. One hypothesis regarding the mechanism by which the APOE4 allele might increase AD risk is loss of adaptive function, raising the possibility that the exogenous administration of apoE mimetics would have therapeutic effects. In this study, we utilized a previously characterized murine model of AD containing human APP, PS1 and APOE4TR, the APP/PS1/APOETR mouse. We treated male APP/PS1/APOETR mice with the apoE mimetic CN-105 or vehicle for 40d, beginning either at 14-18 or 25-28 weeks of age. After termination of treatment we tested animals in both Morris water maze and contextual fear conditioning, and examined soluble Aß by biochemistery and Aß deposition in cortex by unbiased stereology. We found that transient treatment with CN-105 for 40d beginning at 14-18 weeks reduced Aß pathology and rescued memory deficits in male APP/PS1/APOETR mice. Notably, delaying treatment onset to 25-28 weeks did not produce as robust an effect. These results suggest CN-105 treatment in a mouse model of AD results in a reduction in AD pathology and improved behavioral outcomes when administered early in the course of disease. As CN-105 has an excellent safety profile and is already in clinical trials, these findings raise the possibility that CN-105 represents a novel and translatable therapeutic strategy for AD.

Apolipoprotein E Peptide-Guided Disulfide-Cross-Linked Micelles for Targeted Delivery of Sorafenib to Hepatocellular Carcinoma.

Sorafenib (SF) is an FDA-approved molecular-targeted drug for treating hepatocellular carcinoma (HCC). SF, however, suffers from poor water solubility, low bioavailability, dose-limiting side effects, and possible drug resistance. Here, we report on apolipoprotein E peptide-decorated disulfide-cross-linked micellar SF (ApoE-Ms-SF) as a targeted and intelligent formulation for HCC therapy. ApoE-Ms-SF was prepared with a good SF loading of 7.0 wt %, small size (37 nm), high stability, and reduction-triggered drug release from poly(ethylene glycol)-b-poly(ε-caprolactone-co-dithiolane trimethylene carbonate)-mefenamate (PEG-P(CL-DTC)-MA) and ApoE-modified ApoE-PEG-P(CL-DTC) block copolymers. MTT assays in low-density lipoprotein receptors (LDLRs) overexpressing SMMC-7721 human liver cancer cells showed ApoE density-dependent antitumor potency of ApoE-Ms-SF, in which 7.5% ApoE led to the best antitumor effect (IC50: 8.5 vs 23.3 µg/mL for free SF). Confocal studies, flow cytometry, western blot, and apoptotic assays illustrated clearly a more efficient uptake of ApoE-Ms than nontargeted Ms by SMMC-7721 cells as well as lower phosphorylated extracellular signal-regulated kinase protein level and better cell apoptosis caused by ApoE-Ms-SF compared with Ms-SF and free SF. ApoE-Ms-SF revealed a long circulation time (elimination half-life = 6.8 h). DiR-loaded ApoE-Ms showed a significantly higher accumulation in SMMC-7721 tumor than the nontargeted counterpart. The therapeutic outcomes in the orthotopic SMMC-7721 tumor models demonstrated that ApoE-Ms-SF reduced SF-associated side effects and brought about enhanced angiogenesis inhibition and tumor apoptosis compared to free SF and Ms-SF controls, leading to a better treatment of HCC.

Vitamin B3 impairs reverse cholesterol transport in Apolipoprotein E-deficient mice / La vitamina B3 altera el transporte reverso del colesterol en ratones con deficiencia en apolipoproteína E

Introduction: High Density Lipoproteins (HDL) are dysfunctional in hypercholesterolemia patients. The hypothesis was tested that nicotinamide (NAM) administration will influence HDL metabolism and reverse cholesterol transport from macrophages to the liver and feces in vivo (m-RCT) in a murine model of hypercholesterolemia. Methods: Apolipoprotein E-deficient (KOE) mice were challenged with a high-fat diet for 4 weeks. The effect of different doses of NAM on cholesterol metabolism, and the ability of HDL to promote m-RCT was assessed. Results: The administration of NAM to KOE mice produced an increase (∼1.5-fold; P < 0.05) in the plasma levels of cholesterol, which was mainly accounted for by the non-HDL fraction. NAM produced a [3H]-cholesterol plasma accumulation (∼1.5-fold) in the m-RCT setting. As revealed by kinetic analysis, the latter was mainly explained by an impaired clearance of circulating non-HDL (∼0.8-fold). The relative content of [3H]-tracer was lowered in the livers (∼0.6-fold) and feces (> 0.5-fold) of NAM-treated mice. This finding was accompanied by a significant (or trend close to significance) up-regulation of the relative gene expression of Abcg5 and Abcg8 in the liver (Abcg5: 2.9-fold; P < 0.05; Abcg8: 2.4-fold; P = 0.06) and small intestine (Abcg5: 2.1-fold; P = 0.15; Abcg8: 1.9-fold; P < 0.05) of high-dose, NAM-treated mice. Conclusion: The data from this study show that the administration of NAM to KOE mice impaired m-RCT in vivo. This finding was partly due to a defective hepatic clearance of plasma non-HDL

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The Dynamics of Circulating Monocyte Subsets and Intra-Plaque Proliferating Macrophages during the Development of Atherosclerosis in ApoE-/- Mice.

To detect the development of monocytes and proliferative macrophages in atherosclerosis of ApoE-/- mice, we randomly assigned 84 ApoE-/- mice fed western diet or chow diet. On weeks 2, 4, 6, 8, 10, and 12 after fed high-fat diet or normal chow diet, animals were euthanized (n = 7 for each group at each time point). Flow cytometry methods were used to analyze the proportions of circulation monocyte subsets. The macrophage and proliferative macrophage accumulation within atherosclerotic plaques was estimated by confocal florescence microscopy. Plasma levels of total cholesterol and triglyceride were measured by ELISA kit. The plaques of aortic sinus were stained with Oil Red O. The percent of Ly6Chi circulation monocyte, the density of proliferation macrophage, the total plasma cholesterol and triglyceride levels, the lesion area of ApoE-/- mice were consistently elevated in chow diet throughout the trial. The total plasma cholesterol and triglyceride levels, the lesion area were elevated in western diet group with age, and they were always higher than the chow diet group. The Ly6Chi monocytes and proliferative macrophages reached a plateau at 8 weeks and 6 weeks; despite continued high-triglyceride high-cholesterol diet the percent did not significantly change. Interestingly, the density of macrophage did not change significantly over age in western and chow diet groups. Our results provide a dynamic view of Ly6Chi monocyte subset, the density of macrophage and proliferation macrophage change during the development and progression of atherosclerosis, which is relevant for designing new treatment strategies targeting mononuclear phagocytes in this model.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy.

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ±â€¯1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

Administration of Downstream ApoE Attenuates the Adverse Effect of Brain ABCA1 Deficiency on Stroke.

The ATP-binding cassette transporter member A1 (ABCA1) and apolipoprotein E (ApoE) are major cholesterol transporters that play important roles in cholesterol homeostasis in the brain. Previous research demonstrated that specific deletion of brain-ABCA1 (ABCA1-B/-B) reduced brain grey matter (GM) and white matter (WM) density in the ischemic brain and decreased functional outcomes after stroke. However, the downstream molecular mechanism underlying brain ABCA1-deficiency-induced deficits after stroke is not fully understood. Adult male ABCA1-B/-B and ABCA1-floxed control mice were subjected to distal middle-cerebral artery occlusion and were intraventricularly infused with artificial mouse cerebrospinal fluid as vehicle control or recombinant human ApoE2 into the ischemic brain starting 24 h after stroke for 14 days. The ApoE/apolipoprotein E receptor 2 (ApoER2)/high-density lipoprotein (HDL) levels and GM/WM remodeling and functional outcome were measured. Although ApoE2 increased brain ApoE/HDL levels and GM/WM density, negligible functional improvement was observed in ABCA1-floxed-stroke mice. ApoE2-administered ABCA1-B/-B stroke mice exhibited elevated levels of brain ApoE/ApoER2/HDL, increased GM/WM density, and neurogenesis in both the ischemic ipsilateral and contralateral brain, as well as improved neurological function compared with the vehicle-control ABCA1-B/-B stroke mice 14 days after stroke. Ischemic lesion volume was not significantly different between the two groups. In vitro supplementation of ApoE2 into primary cortical neurons and primary oligodendrocyte-progenitor cells (OPCs) significantly increased ApoER2 expression and enhanced cholesterol uptake. ApoE2 promoted neurite outgrowth after oxygen-glucose deprivation and axonal outgrowth of neurons, and increased proliferation/survival of OPCs derived from ABCA1-B/-B mice. Our data indicate that administration of ApoE2 minimizes the adverse effects of ABCA1 deficiency after stroke, at least partially by promoting cholesterol traffic/redistribution and GM/WM remodeling via increasing the ApoE/HDL/ApoER2 signaling pathway.

Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aß scFv in 3xTg-AD mice.

Anti-Aß immunotherapy has emerged as a promising approach to treat Alzheimer's disease (AD). The single-chain variable fragment scFv-h3D6 is an anti-Aß antibody fragment that lacks the Fc region, which is associated with the induction of microglial reactivity by the full-length monoclonal antibody bapineuzumab. ScFv-h3D6 was previously shown to restore the levels of apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a triple-transgenic-AD (3xTg-AD) mouse model. Since apoE and apoJ play an important role in the development of AD, we aimed to study the in vivo effect of the combined therapy of scFv-h3D6 with apoE and apoJ mimetic peptides (MPs). Four-and-a-half-month-old 3xTg-AD mice were treated for six weeks with scFv-h3D6, apoE-MP, apoJ-MP, or a combination of scFv-h3D6 with each of the MPs, or a vehicle, and then the results were compared to non-transgenic mice. Magnetic Resonance Imaging showed a general tendency of the different treatments to protect against the reduction in brain volume. Aß burden decreased after treatment with scFv-h3D6, apoE-MP, or apoJ-MP, but the effect was not as evident with the combined therapies. In terms of glial reactivity, apoE-MP showed a potent anti-inflammatory effect that was eased by the presence of scFv-h3D6, whereas the combination of apoJ-MP and scFv-h3D6 was not detrimental. ScFv-h3D6 alone did not induce microglial reactivity, as full-length antibodies do; rather, it reduced it. Endogenous apoE and apoJ levels were decreased by scFv-h3D6, but the MPs lead to a simultaneous increase of both apolipoproteins. While apoE-MP and apoJ-MP demonstrated different effects in the combined therapies with scFv-h3D6, they did not improve the overall protective effect of scFv-h3D6 in reducing the Aß burden, apolipoproteins levels or microglial reactivity.

Amphiphilic lipopeptide significantly enhances uptake of charge-neutral splice switching morpholino oligonucleotide in spinal muscular atrophy patient-derived fibroblasts.

Splice-switching antisense oligonucleotides (SSOs) are emerging therapeutics with two SSOs recently approved by the FDA for Duchenne muscular dystrophy and spinal muscular atrophy. SSOs are administered without any delivery vector and require large doses to achieve the therapeutic benefit, primarily due to their poor cellular uptake. Although cell-penetrating peptides (CPP) have shown great potential in delivering SSOs into cells, their capacity as delivery vector is limited. Here we have studied the effect of lipid conjugation on the cell permeability of a known CPP (ApoE). Myristic acid was coupled at the N-terminus of ApoE to a C-terminal cysteine residue. The myristoylated ApoE (Myr-ApoE) was conjugated to a maleimide functionalised phosphorodiamidate morpholino oligonucleotide (PMO). The Myr-ApoE-PMO conjugate showed no cytoxicity and had significantly higher efficiency in cell permeability with 30% higher splice-switching activity compared to ApoE-PMO. The self-assembly properties of this amphiphilic lipopeptide-PMO conjugate was assessed. Transmission electron microscopy showed formation of nanoparticles with amphiphile behaviour and spherical structure. The self-assembly of Myr-ApoE-PMO into nanoparticles enabled it to better bind to cell membranes and to be more efficiently taken up by fibroblast cells. These results showed that modification of physico-chemical properties of peptides to produce peptide amphiphiles enhances cellular uptake and can be used as an efficient delivery vector for therapeutic SSOs.

Multifunctional liposomes delay phenotype progression and prevent memory impairment in a presymptomatic stage mouse model of Alzheimer disease.

The failure of clinical trials largely focused on mild to moderate stages of Alzheimer disease has suggested to the scientific community that the effectiveness of Amyloid-ß (Aß)-centered treatments should be evaluated starting as early as possible, well before irreversible brain damage has occurred. Accordingly, also the preclinical development of new therapies should be carried out taking into account this suggestion. In the present investigation we evaluated the efficacy of a treatment with liposomes multifunctionalized for crossing the blood-brain barrier and targeting Aß, carried out on young APP/PS1 Tg mice, taken as a model of pre-symptomatic disease stage. Liposomes were administered once a week to Tg mice for 7months, starting at the age of 5months and up to the age of 12 when they display AD-like cognitive and brain biochemical/anatomical features. The treatment prevented the onset of the long-term memory impairment and slowed down the deposition of brain Aß; at anatomical level, prevented both ventricle enlargement and entorhinal cortex thickness reduction, otherwise occurring in untreated mice. Strikingly, these effects were maintained 3months after treatment discontinuation. An increase of Aß levels in the liver was detected at the end of the treatment, then followed also by reduction of brain Amyloid Precursor Protein and increase of Aß-degrading enzymes. These results suggest that the treatment promotes brain Aß clearance by a peripheral 'sink' effect and ultimately affects Aß turnover in the brain. Worth of note, the treatment was apparently not toxic for all the organs analyzed, in particular for brain, as suggested by the lower brain TNF-α and MDA levels, and by higher level of SOD activity in treated mice. Together, these findings promote a very early treatment with multi-functional liposomes as a well-tolerated nanomedicine-based approach, potentially suitable for a disease-modifying therapy of AD, able to delay or prevent relevant features of the disease.

Apolipoprotein E Isoforms Differentially Regulate Alzheimer's Disease and Amyloid-ß-Induced Inflammatory Response in vivo and in vitro.

Neuroinflammation plays a critical role in neuronal dysfunction and death of Alzheimer's disease (AD). ApoE4 is a major risk factor of AD, while ApoE2 is neuroprotective. Little is known about the roles of ApoE isoforms in the neuroinflammation seen in AD. Their roles and mechanisms in Aß-induced/neuroinflammation were investigated in this study using in vivo and in vitro models. Rat astrocytes were treated with lipid-poor recombinant hApoE and/or Aß42. Mouse astrocyte lines-expressing lipidated hApoE were treated with Aß42 and/or vitamin D receptor (VDR) agonist, 1α,25-dihydroxyvitamin D3. Cells and media were harvested for cytokine ELISA, RNA isolated for qRT-PCR, and nuclear protein for transcription factor (TF) arrays and EMSA. hApoE-transgenic and AD mice were mated to generate hApoE2/AD and hApoE4/AD mice. Mice were euthanized at 6 months of age. Brain tissues were collected for cytokine ELISA array, Aß ELISA, immunoblotting, and immunohistochemistry. hApoE4/AD mice had significantly higher levels of inflammatory cytokines than hApoE2/AD mice. Lipidated hApoE4 significantly promoted inflammatory gene expression induced by Aß42 but not recombinant hApoE4 in astrocytes as compared to controls. Lipidated hApoE3 provided a certain degree of protection against Aß42-induced inflammatory response but not recombinant hApoE3 as compared to controls. Both lipidated and recombinant hApoE2 provided protection against Aß42-induced inflammatory response compared to controls. TF array revealed that ApoE2 strongly activated VDR in Aß42-treated astrocytes. Application of 1α,25-dihydroxyvitamin D3 completely inhibited Aß-induced inflammatory gene expression in hApoE4-expressing astrocytes. The results suggest that ApoE4 promotes, but ApoE2 inhibits, AD/Aß-induced neuroinflammation via VDR signaling. Targeting VDR signaling or active form of VD3 may relieve AD neuroinflammation or/and neurodegeneration.

Apolipoprotein E Mimetic Peptide Increases Cerebral Glucose Uptake by Reducing Blood-Brain Barrier Disruption after Controlled Cortical Impact in Mice: An 18F-Fluorodeoxyglucose PET/CT Study.

Traumatic brain injury (TBI) disrupts the blood-brain barrier (BBB) and reduces cerebral glucose uptake. Vascular endothelial growth factor (VEGF) is believed to play a key role in TBI, and COG1410 has demonstrated neuroprotective activity in several models of TBI. However, the effects of COG1410 on VEGF and glucose metabolism following TBI are unknown. The current study aimed to investigate the expression of VEGF and glucose metabolism effects in C57BL/6J male mice subjected to experimental TBI. The results showed that controlled cortical impact (CCI)-induced vestibulomotor deficits were accompanied by increases in brain edema and the expression of VEGF, with a decrease in cerebral glucose uptake. COG1410 treatment significantly improved vestibulomotor deficits and glucose uptake and produced decreases in VEGF in the pericontusion and ipsilateral hemisphere of injury, as well as in brain edema and neuronal degeneration compared with the control group. These data support that COG1410 may have potential as an effective drug therapy for TBI.

Biomimetic ApoE-Reconstituted High Density Lipoprotein Nanocarrier for Blood-Brain Barrier Penetration and Amyloid Beta-Targeting Drug Delivery.

Amyloid beta (Aß) and its aggregation forms in the brain have been suggested as key targets for the therapy of Alzheimer's disease (AD). Therefore, the development of nanocarriers that possess both blood-brain barrier permeability and Aß-targeting ability is of great importance for the intervention of AD. Here we constructed a biomimetic nanocarrier named apolipoprotein E (ApoE)-reconstituted high density lipoprotein nanocarrier (ANC) from recombinant ApoE and synthetic lipids to achieve the above goals. α-Mangostin (α-M), a polyphenolic agent that can inhibit the formation of Aß oligomers and fibrils and accelerate Aß cellular degradation, was used as the model drug. Compared with the control liposome, ANC demonstrated about 54-fold higher cellular uptake in brain endothelial cell line in vitro in an ApoE-dependent manner and much higher brain delivery efficiency in vivo. Confocal microscopy analysis witnessed the penetration of ANC across the brain vessels and its accumulation at the surrounding of Aß aggregates. Following the loading of α-M, the Aß-binding affinity of the nanoformulation (ANC-α-M) was not reduced but even enhanced. The effect of ANC-α-M on facilitating the microglia-mediated uptake and degradation of Aß1-42 was enhanced by 336% and 29-fold when compared with that of the nontreated control and also much higher than that of ANC. Following intravenous administration for 2 to 4 weeks, ANC-α-M exhibited the most efficient efficacy in decreasing amyloid deposition, attenuating microgliosis, and rescuing memory defect in SAMP8 mice, an AD mouse model. Taken together, the findings of this work provided strong evidence that the ApoE-based biomimetic nanocarrier could provide a promising platform for brain drug delivery toward the treatment of AD.

An apoE-derived mimic peptide, COG1410, alleviates early brain injury via reducing apoptosis and neuroinflammation in a mouse model of subarachnoid hemorrhage.

This study investigated the neuroprotective effects of COG1410, an apoliporotein E (apoE)-derived mimic peptide, against early brain injury (EBI) after subarachnoid hemorrhage (SAH). SAH was induced in C57BL/6J mice (n=68) by endovascular perforation. Mice received intravenous injection of COG1410 (2mg/kg) or equal volume of vehicle (saline). The mortality rate, neurological score, rotarod latencies, cell apoptosis, microglial activation, pro-inflammatory cytokines production and protein levels of apoptotic and inflammatory markers were assessed at 24h after sham operation or SAH. Results showed that COG1410 alleviated the neurological deficits associated with SAH. Compared with vehicle treatment group, the number of apoptotic cells and activated microglia decreased significantly in the COG1410 treated group. COG1410 enhanced Akt activation and suppressed caspase-3 cleavage. The imbalance of Bax and Bcl-2 induced by SAH was regulated by COG1410. Additionally, COG1410 attenuated cytokines production of IL-1ß, IL-6 and TNF-α and suppressed the activation of JNK/c-Jun and NF-κB. Taken together, COG1410 protected against EBI via reducing apoptosis and neuroinflammation, through mechanisms that involve the regulation of apoptotic signaling and microglial activation. COG1410 is a potential neuroprotective agent for SAH treatment.

A new cryptic cationic antimicrobial peptide from human apolipoprotein E with antibacterial activity and immunomodulatory effects on human cells.

Cationic antimicrobial peptides (AMPs) possess fast and broad-spectrum activity against both Gram-negative and Gram-positive bacteria, as well as fungi. It has become increasingly evident that many AMPs, including those that derive from fragments of host proteins, are multifunctional and able to mediate various immunomodulatory functions and angiogenesis. Among these, synthetic apolipoprotein-derived peptides are safe and well tolerated in humans and have emerged as promising candidates in the treatment of various inflammatory conditions. Here, we report the characterization of a new AMP corresponding to residues 133-150 of human apolipoprotein E. Our results show that this peptide, produced either by chemical synthesis or by recombinant techniques in Escherichia coli, possesses a broad-spectrum antibacterial activity. As shown for several other AMPs, ApoE (133-150) is structured in the presence of TFE and of membrane-mimicking agents, like SDS, or bacterial surface lipopolysaccharide (LPS), and an anionic polysaccharide, alginate, which mimics anionic capsular exo-polysaccharides of several pathogenic microorganisms. Noteworthy, ApoE (133-150) is not toxic toward several human cell lines and triggers a significant innate immune response, assessed either as decreased expression levels of proinflammatory cytokines in differentiated THP-1 monocytic cells or by the induction of chemokines released from PBMCs. This novel bioactive AMP also showed a significant anti-inflammatory effect on human keratinocytes, suggesting its potential use as a model for designing new immunomodulatory therapeutics.

Gene Therapy Strategies for Alzheimer's Disease: An Overview.

Key neuropathological hallmarks of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular accumulation of hyperphosphorylated Tau protein. The mechanisms underlying these neuropathological changes remain unclear. So far, research on AD therapy has had limited success in terms of symptomatic treatments although it has also had several failures for disease-modifying drugs. Gene transfer strategies to the brain have contributed to evaluate in animal models many interesting tracks, some of which should deserve clinical applications in AD patients in the future.