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1.
Commun Biol ; 4(1): 61, 2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420340

RESUMO

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

2.
Neuron ; 109(3): 438-447.e6, 2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33321072

RESUMO

The ε4 allele of the apolipoprotein E gene (APOE4) is a strong genetic risk factor for Alzheimer's disease (AD) and multiple vascular conditions. ApoE is abundantly expressed in multiple brain cell types, including astrocytes, microglia, and vascular mural cells (VMCs). Here, we show that VMC-specific expression of apoE4 in mice impairs behavior and cerebrovascular function. Expression of either apoE3 or apoE4 in VMCs was sufficient to rescue the hypercholesterolemia and atherosclerosis phenotypes seen in Apoe knockout mice. Intriguingly, vascular expression of apoE4, but not apoE3, reduced arteriole blood flow, impaired spatial learning, and increased anxiety-like phenotypes. Single-cell RNA sequencing of vascular and glial cells revealed that apoE4 in VMCs was associated with astrocyte activation, while apoE3 was linked to angiogenic signature in pericytes. Together, our data support cell-autonomous effects of vascular apoE on brain homeostasis in an isoform-dependent manner, suggesting a critical contribution of vascular apoE to AD pathogenesis.

3.
J Alzheimers Dis ; 2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33285637

RESUMO

BACKGROUND: Accumulation of hyperphosphorylated tau (pTau) protein is associated with synaptic dysfunction in Alzheimer's disease (AD). We previously demonstrated that neuroprotection in familial mouse models of AD could be achieved by targeting mitochondria complex I (MCI) and activating the adaptive stress response. Efficacy of this strategy on pTau-related pathology remained unknown. OBJECTIVE: To investigate the effect of specific MCI inhibitor tricyclic pyrone compound CP2 on levels of human pTau, memory function, long term potentiation (LTP), and energy homeostasis in 18-month-old 3xTg-AD mice and explore the potential mechanisms. METHODS: CP2 was administered to male and female 3xTg-AD mice from 3.5-18 months of age. Cognitive function was assessed using the Morris water maze. Glucose metabolism was measured in periphery using a glucose tolerance test and in the brain using fluorodeoxyglucose F18 positron-emission tomography (FDG-PET). LTP was evaluated using electrophysiology in the hippocampus. The expression of key proteins associated with neuroprotective mechanisms were assessed by western blotting. RESULTS: Chronic CP2 treatment restored synaptic activity in female 3xTg-AD mice; cognitive function, levels of synaptic proteins, glucose metabolism, and energy homeostasis were improved in male and female 3xTg-AD mice. Significant reduction of human pTau in the brain was associated with increased activity of protein phosphatase of type 2A (PP2A), and reduced activity of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3ß (GSK3ß). CONCLUSION: CP2 treatment protected against synaptic dysfunction and memory impairment in symptomatic 3xTg-AD mice, and reduced levels of human pTau, indicating that targeting mitochondria with small molecule specific MCI inhibitors represents a promising strategy for treating AD.

4.
Nat Commun ; 11(1): 5540, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139712

RESUMO

APOE4 is the strongest genetic risk factor associated with late-onset Alzheimer's disease (AD). To address the underlying mechanism, we develop cerebral organoid models using induced pluripotent stem cells (iPSCs) with APOE ε3/ε3 or ε4/ε4 genotype from individuals with either normal cognition or AD dementia. Cerebral organoids from AD patients carrying APOE ε4/ε4 show greater apoptosis and decreased synaptic integrity. While AD patient-derived cerebral organoids have increased levels of Aß and phosphorylated tau compared to healthy subject-derived cerebral organoids, APOE4 exacerbates tau pathology in both healthy subject-derived and AD patient-derived organoids. Transcriptomics analysis by RNA-sequencing reveals that cerebral organoids from AD patients are associated with an enhancement of stress granules and disrupted RNA metabolism. Importantly, isogenic conversion of APOE4 to APOE3 attenuates the APOE4-related phenotypes in cerebral organoids from AD patients. Together, our study using human iPSC-organoids recapitulates APOE4-related phenotypes and suggests APOE4-related degenerative pathways contributing to AD pathogenesis.


Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Regulação da Expressão Gênica , Genótipo , Humanos , Organoides/patologia , RNA/metabolismo , Transcriptoma
5.
Elife ; 92020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33074098

RESUMO

Although the ε2 allele of apolipoprotein E (APOE2) benefits longevity, its mechanism is not understood. The protective effects of the APOE2 on Alzheimer's disease (AD) risk, particularly through their effects on amyloid or tau accumulation, may confound APOE2 effects on longevity. Herein, we showed that the association between APOE2 and longer lifespan persisted irrespective of AD status, including its neuropathology, by analyzing clinical datasets as well as animal models. Notably, APOE2 was associated with preserved activity during aging, which also associated with lifespan. In animal models, distinct apoE isoform levels, where APOE2 has the highest, were correlated with activity levels, while some forms of cholesterol and triglycerides were associated with apoE and activity levels. These results indicate that APOE2 can contribute to longevity independent of AD. Preserved activity would be an early-observable feature of APOE2-mediated longevity, where higher levels of apoE2 and its-associated lipid metabolism might be involved.

6.
Alzheimers Dement ; 16(10): 1372-1383, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32827351

RESUMO

INTRODUCTION: Cerebrovascular pathologies including cerebral amyloid angiopathy (CAA) and blood-brain barrier (BBB) dysregulation are prominent features in the majority of Alzheimer's disease (AD) cases. METHODS: We performed neuropathologic and biochemical studies on a large, neuropathologically confirmed human AD cohort (N = 469). Amounts of endothelial tight junction proteins claudin-5 (CLDN5) and occludin (OCLN), and major AD-related molecules (amyloid beta [Aß40], Aß42, tau, p-tau, and apolipoprotein E) in the temporal cortex were assessed by ELISA. RESULTS: Higher levels of soluble tau, insoluble p-tau, and apolipoprotein E (apoE) were independently correlated with lower levels of endothelial tight junction proteins CLDN5 and OCLN in AD brains. Although high Aß40 levels, APOE ε4, and male sex were predominantly associated with exacerbated CAA severity, those factors did not influence tight junction protein levels. DISCUSSION: Refining the molecular mechanisms connecting tau, Aß, and apoE with cerebrovascular pathologies is critical for greater understanding of AD pathogenesis and establishing effective therapeutic interventions for the disease.

7.
Nanomedicine ; 28: 102225, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32485318

RESUMO

The limitations imposed on brain therapy by the blood-brain barrier (BBB) have warranted the development of carriers that can overcome and deliver therapeutic agents into the brain. We strategically designed liposomal nanoparticles encasing plasmid DNA for efficient transfection and translocation across the in vitro BBB model as well as in vivo brain-targeted delivery. Liposomes were surface modified with two ligands, cell-penetrating peptide (PFVYLI or R9F2) for enhanced internalization into cells and transferrin (Tf) ligand for targeting transferrin-receptor expressed on brain capillary endothelial cells. Dual-modified liposomes encapsulating pDNA demonstrated significantly (P < 0.05) higher in vitro transfection efficiency compared to single-modified nanoparticles. R9F2Tf-liposomes showed superior ability to cross in vitro BBB and, subsequently, transfect primary neurons. Additionally, these nanoparticles crossed in vivo BBB and reached brain parenchyma of mice (6.6%) without causing tissue damage. Transferrin receptor-targeting with enhanced cell penetration is a relevant strategy for efficient brain-targeted delivery of genes.

8.
J Pharmacol Exp Ther ; 374(3): 354-365, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32561686

RESUMO

The development of neuropharmaceutical gene delivery systems requires strategies to obtain efficient and effective brain targeting as well as blood-brain barrier (BBB) permeability. A brain-targeted gene delivery system based on a transferrin (Tf) and cell-penetrating peptide (CPP) dual-functionalized liposome, CPP-Tf-liposome, was designed and investigated for crossing BBB and permeating into the brain. We selected three sequences of CPPs [melittin, Kaposi fibroblast growth factor (kFGF), and penetration accelerating sequence-R8] and compared their ability to internalize into the cells and, subsequently, improve the transfection efficiency. Study of intracellular uptake indicated that liposomal penetration into bEnd.3 cells, primary astrocytes, and primary neurons occurred through multiple endocytosis pathways and surface modification with Tf and CPP enhanced the transfection efficiency of the nanoparticles. A coculture in vitro BBB model reproducing the in vivo anatomophysiological complexity of the biologic barrier was developed to characterize the penetrating properties of these designed liposomes. The dual-functionalized liposomes effectively crossed the in vitro barrier model followed by transfecting primary neurons. Liposome tissue distribution in vivo indicated superior ability of kFGF-Tf-liposomes to overcome BBB and reach brain of the mice after single intravenous administration. These findings demonstrate the feasibility of using strategically designed liposomes by combining Tf receptor targeting with enhanced cell penetration as a potential brain gene delivery vector. SIGNIFICANCE STATEMENT: Rational synthesis of efficient brain-targeted gene carrier included modification of liposomes with a target-specific ligand, transferrin, and with cell-penetrating peptide to enhance cellular internalization. Our study used an in vitro triple coculture blood-brain barrier (BBB) model as a tool to characterize the permeability across BBB and functionality of designed liposomes prior to in vivo biodistribution studies. Our study demonstrated that rational design and characterization of BBB permeability are efficient strategies for development of brain-targeted gene carriers.


Assuntos
Encéfalo/efeitos dos fármacos , Lipossomos/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Feminino , Técnicas de Transferência de Genes , Terapia Genética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos Sprague-Dawley , Distribuição Tecidual/fisiologia , Transferrina/administração & dosagem
9.
Mol Pharm ; 17(6): 2054-2063, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32315185

RESUMO

The therapeutic potential of the nerve growth factor (NGF) gene using brain-targeted liposomal nanoparticles was investigated for the treatment of Alzheimer's disease (AD). We designed brain-targeted gene delivery systems with prolonged systemic circulation and enhanced cellular penetration by conjugating the transferrin (Tf) ligand and the penetratin (Pen) peptide to liposomes via a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) phospholipid. In vitro characterization studies showed that the nanoparticles had homogeneous particle size and positive zeta potential and were able to protect the plasmid DNA against enzymatic degradation. In vivo brain targeting efficiency of designed liposomes was mimicked using an in vitro triple coculture blood-brain barrier (BBB) model. Liposomal nanoparticles dual-modified with Tf and Pen encasing plasmid NGF efficiently crossed the in vitro BBB model and, subsequently, transfected the primary neuronal cells. Increasing NGF expression in primary neuronal cells following treatment with liposomes increased the levels of the presynaptic marker synaptophysin in vitro. A dose-response study in vivo was performed in order to select the appropriate dose of plasmid NGF to induce significant NGF expression and, consequently, a therapeutic effect. Administration of PenTf-liposomes containing pNGF to amyloid precursor protein (APP)/PS1 mice (aged 3 months) for 4 weeks (one injection per week) significantly decreased (p < 0.05) the levels of toxic soluble and insoluble Aß peptides as compared to those levels in untreated APP/PS1 mice. Additionally, the treatment stimulated cell proliferation and increased the levels of synaptic markers, synaptophysin and PSD-95. These data suggest the therapeutic potential of PenTf-liposome-mediated NGF gene therapy, and this system can be considered a candidate for the treatment of AD.

10.
Neuron ; 106(5): 727-742.e6, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32199103

RESUMO

Evidence suggests interplay among the three major risk factors for Alzheimer's disease (AD): age, APOE genotype, and sex. Here, we present comprehensive datasets and analyses of brain transcriptomes and blood metabolomes from human apoE2-, apoE3-, and apoE4-targeted replacement mice across young, middle, and old ages with both sexes. We found that age had the greatest impact on brain transcriptomes highlighted by an immune module led by Trem2 and Tyrobp, whereas APOE4 was associated with upregulation of multiple Serpina3 genes. Importantly, these networks and gene expression changes were mostly conserved in human brains. Finally, we observed a significant interaction between age, APOE genotype, and sex on unfolded protein response pathway. In the periphery, APOE2 drove distinct blood metabolome profile highlighted by the upregulation of lipid metabolites. Our work identifies unique and interactive molecular pathways underlying AD risk factors providing valuable resources for discovery and validation research in model systems and humans.


Assuntos
Envelhecimento/genética , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Encéfalo/metabolismo , Serpinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Fatores Etários , Doença de Alzheimer/metabolismo , Animais , Apolipoproteína E2/genética , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Genótipo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Metaboloma , Camundongos , Camundongos Transgênicos , Fatores de Proteção , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Fatores de Risco , Fatores Sexuais , Resposta a Proteínas não Dobradas/genética
11.
Brain Res ; 1734: 146738, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32081534

RESUMO

Effective transport of therapeutic nucleic acid to brain has been a challenge for the success of gene therapy for treating brain diseases. In this study, we proposed liposomal nanoparticles modified with brain targeting ligandsfor active brain targeting with enhanced BBB permeation and delivery of genes to brain. We targeted transferrin and nicotinic acetylcholine receptors by conjugating transferrin (Tf) and rabies virus glycoprotein (RVG) peptide to surface of liposomes. Liposomal formulations showed homogeneous particle size and ability to protect plasmid DNA against enzymatic degradation. These nanoparticles were internalized by brain endothelial cells, astrocytes and primary neuronal cells through energy-dependent endocytosis pathways. RVG-Tf coupled liposomes showed superior ability to transfect cells compared to liposomes without surface modification or single modification. Characterization of permeability through blood brain barrier (BBB) and functionality of designed liposomes were performed using an in vitro triple co-culture BBB model. Liposome-RVG-Tf efficiently translocated across in vitro BBB model and, consecutively, transfected primary neuronal cells. Notably, brain-targeted liposomes promoted in vivo BBB permeation. These studies suggest that modifications of liposomes with brain-targeting ligands are a promising strategy for delivery of genes to brain.

12.
J Stroke Cerebrovasc Dis ; 29(4): 104631, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31964576

RESUMO

Background Recent studies of patients with intracerebral hemorrhage suggest an association between peripheral blood neutrophil-lymphocyte ratio and neurologic deterioration. We aimed to study the prognostic utility of neutrophil-lymphocyte ratio in predicting inpatient mortality in aneurysmal subarachnoid hemorrhage. Methods We conducted a retrospective electronic medical record review of the clinical, laboratory, and radiographic data of patients with aneurysmal subarachnoid hemorrhage 18 years of age or older presenting to the neuroscience intensive care unit from January 1, 2011, to December 31, 2017. Patients with aneurysmal subarachnoid hemorrhage were divided into 2 groups (group 1, alive at discharge; group 2, deceased prior to discharge), and neutrophil-lymphocyte ratio laboratory mean values were recorded for each patient. Our primary outcome measure was inpatient mortality, and our secondary measure was incidence of pneumonia with hospitalization. Results We identified 403 patients with aneurysmal subarachnoid hemorrhage for the study. After exclusion criteria, 44 eligible patients were divided into the 2 groups (group 1, n = 32; group 2, n = 12). Mean neutrophil-lymphocyte ratio for group 1 was 11.53, and for group 2, 17.85 (P < .01). The mean neutrophil-lymphocyte ratio of those who developed pneumonia compared to those who did not was 15.28 versus 12.81, respectively (P = .39). A Kaplan-Meier plot demonstrated increased mortality among patients with a neutrophil-lymphocyte ratio equal to or greater than 12.5 compared to those with a neutrophil-lymphocyte ratio less than 12.5. Conclusions These preliminary data demonstrate that a neutrophil-lymphocyte ratio equal to or greater than 12.5 at admission predict higher inpatient mortality in patients with aneurysmal subarachnoid hemorrhage.


Assuntos
Linfócitos/imunologia , Neutrófilos/imunologia , Hemorragia Subaracnóidea/imunologia , Adulto , Idoso , Registros Eletrônicos de Saúde , Feminino , Mortalidade Hospitalar , Humanos , Incidência , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Admissão do Paciente , Readmissão do Paciente , Pneumonia/imunologia , Pneumonia/mortalidade , Pneumonia/terapia , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Hemorragia Subaracnóidea/sangue , Hemorragia Subaracnóidea/mortalidade , Hemorragia Subaracnóidea/terapia
13.
Arterioscler Thromb Vasc Biol ; 40(1): 128-144, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31665905

RESUMO

OBJECTIVE: The ε4 allele of the APOE gene (APOE4) is the strongest genetic risk factor for Alzheimer disease when compared with the common ε3 allele. Although there has been significant progress in understanding how apoE4 (apolipoprotein E4) drives amyloid pathology, its effects on amyloid-independent pathways, in particular cerebrovascular integrity and function, are less clear. Approach and Results: Here, we show that brain pericytes, the mural cells of the capillary walls, differentially modulate endothelial cell phenotype in an apoE isoform-dependent manner. Extracellular matrix protein induction, tube-like structure formation, and barrier formation were lower with endothelial cells cocultured with pericytes isolated from apoE4-targeted replacement (TR) mice compared with those from apoE3-TR mice. Importantly, aged apoE4-targeted replacement mice had decreased extracellular matrix protein expression and increased plasma protein leakages compared with apoE3-TR mice. CONCLUSIONS: ApoE4 impairs pericyte-mediated basement membrane formation, potentially contributing to the cerebrovascular effects of apoE4.


Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Membrana Basal/metabolismo , Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Pericitos/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Apolipoproteínas E/biossíntese , Membrana Basal/patologia , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/patologia , Isoformas de Proteínas
14.
Proc Natl Acad Sci U S A ; 116(47): 23790-23796, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31690660

RESUMO

Carrying premature termination codons in 1 allele of the ABCA7 gene is associated with an increased risk for Alzheimer's disease (AD). While the primary function of ABCA7 is to regulate the transport of phospholipids and cholesterol, ABCA7 is also involved in maintaining homeostasis of the immune system. Since inflammatory pathways causatively or consequently participate in AD pathogenesis, we studied the effects of Abca7 haplodeficiency in mice on brain immune responses under acute and chronic conditions. When acute inflammation was induced through peripheral lipopolysaccharide injection in control or heterozygous Abca7 knockout mice, partial ABCA7 deficiency diminished proinflammatory responses by impairing CD14 expression in the brain. On breeding to App NL-G-F knockin mice, we observed increased amyloid-ß (Aß) accumulation and abnormal endosomal morphology in microglia. Taken together, our results demonstrate that ABCA7 loss of function may contribute to AD pathogenesis by altering proper microglial responses to acute inflammatory challenges and during the development of amyloid pathology, providing insight into disease mechanisms and possible treatment strategies.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Encéfalo/imunologia , Haploinsuficiência , Microglia/imunologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Perfilação da Expressão Gênica , Imunidade Inata/genética , Camundongos , Camundongos Transgênicos , Transcriptoma
15.
Int J Nanomedicine ; 14: 6497-6517, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31616141

RESUMO

Background: The potential of gene therapy for treatment of neurological disorders can be explored using designed lipid-based nanoparticles such as liposomes, which have demonstrated ability to deliver nucleic acid to brain cells. We synthesized liposomes conjugated to cell-penetrating peptides (CPPs) (vascular endothelial-cadherin-derived peptide [pVec], pentapeptide QLPVM and HIV-1 trans-activating protein [TAT]) and transferrin (Tf) ligand, and examined the influence of surface modifications on the liposome delivery capacity and transfection efficiency of encapsulated plasmid DNA. The design of liposomes was based on targeting molecular recognition of transferrin receptor overexpressed on the blood-brain barrier (BBB) with enhanced internalization ability of CPPs. Methods: CPP-Tf-liposomes were characterized by particle size distribution, zeta potential, protection of encapsulated plasmid DNA, uptake mechanisms and transfection efficiencies. An in vitro triple co-culture BBB model selected the liposomal formulations that were able to cross the in vitro BBB and subsequently, transfect primary neuronal cells. The in vivo biodistribution and biocompatibility of selected formulations were also investigated in mice. Results: Liposomal formulations were able to protect the encapsulated plasmid DNA against enzymatic degradation and presented low hemolytic potential and low cytotoxicity at 100 nM phospholipid concentration. Cellular internalization of nanoparticles occurred via multiple endocytosis pathways. CPP-Tf-conjugated liposomes mediated robust transfection of brain endothelial (bEnd.3), primary glial and primary neuronal cells. Liposomes modified with Tf and TAT demonstrated superior ability to cross the barrier layer and subsequently, transfect neuronal cells compared to other formulations. Quantification of fluorescently labeled liposomes and in vivo imaging demonstrated that this system could efficiently overcome the BBB and penetrate the brain of mice (7.7% penetration of injected dose). Conclusion: In vitro screening platforms are important tools to enhance the success of brain-targeted gene delivery systems. The potential of TAT-Tf-liposomes as efficient brain-targeted gene carriers in vitro and in vivo was suggested to be related to the presence of selected moieties on the nanoparticle surface.


Assuntos
Encéfalo/metabolismo , Peptídeos Penetradores de Células/química , Técnicas de Transferência de Genes , Lipídeos/química , Nanopartículas/química , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Sobrevivência Celular , Endocitose , Feminino , Terapia Genética , Proteínas de Fluorescência Verde/metabolismo , Hemólise , Humanos , Lipossomos , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Neurônios/metabolismo , Plasmídeos/metabolismo , Ratos , Distribuição Tecidual
16.
Pharm Res ; 36(11): 161, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31529284

RESUMO

PURPOSE: Apolipoprotein E2 (ApoE2) gene therapy is a potential disease-modifying therapy for Alzheimer's disease (AD). We investigated the potential of plasmid encoding ApoE2 loaded brain-targeted functionalized-liposomes for treatment of AD. This was achieved via systemic administration of liposomes entrapping therapeutic gene targeting the brain of mice. METHODS: Targeting and transfection efficiency of designed liposomes were determined in bEnd.3, primary glial and primary neuronal cells. The ability of liposomal formulations to translocate across in vitro blood-brain barrier (BBB) and, thereafter, transfect primary neuronal cells was investigated using in vitro triple co-culture BBB model. We quantified ApoE expression in the brain of mice after single intravenous injection of brain-targeted liposomes loaded with plasmid ApoE2. RESULTS: Dual surface modification enhanced the in vitro transfection efficiency of designed liposomes. Successful delivery of therapeutic gene overcoming BBB by Transferrin-Penetratin- modified liposomes was demonstrated both in vitro and in vivo. Significant (p < 0.05) increase in ApoE levels in the brain of mice was observed after intravenous administration of Tf-Pen-liposomes encasing plasmid ApoE2. CONCLUSION: The results indicate that dual-ligand based liposomal gene delivery systems had both enhanced brain targeting and gene delivery efficiencies. Transferrin-Penetratin modified liposomes for delivery of plasmid ApoE2 has great potential for AD treatment.


Assuntos
Apolipoproteína E2/genética , Barreira Hematoencefálica/metabolismo , Terapia Genética , Lipossomos/química , Nanopartículas/química , Doença de Alzheimer/terapia , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Feminino , Humanos , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Transferrina/química , Transferrina/metabolismo
17.
Cells ; 8(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466320

RESUMO

Extracellular vesicles (EVs) contribute to a variety of signaling processes and the overall physiological and pathological states of stem cells and tissues. Human induced pluripotent stem cells (hiPSCs) have unique characteristics that can mimic embryonic tissue development. There is growing interest in the use of EVs derived from hiPSCs as therapeutics, biomarkers, and drug delivery vehicles. However, little is known about the characteristics of EVs secreted by hiPSCs and paracrine signaling during tissue morphogenesis and lineage specification. Methods: In this study, the physical and biological properties of EVs isolated from hiPSC-derived neural progenitors (ectoderm), hiPSC-derived cardiac cells (mesoderm), and the undifferentiated hiPSCs (healthy iPSK3 and Alzheimer's-associated SY-UBH lines) were analyzed. Results: Nanoparticle tracking analysis and electron microscopy results indicate that hiPSC-derived EVs have an average size of 100-250 nm. Immunoblot analyses confirmed the enrichment of exosomal markers Alix, CD63, TSG101, and Hsc70 in the purified EV preparations. MicroRNAs including miR-133, miR-155, miR-221, and miR-34a were differently expressed in the EVs isolated from distinct hiPSC lineages. Treatment of cortical spheroids with hiPSC-EVs in vitro resulted in enhanced cell proliferation (indicated by BrdU+ cells) and axonal growth (indicated by ß-tubulin III staining). Furthermore, hiPSC-derived EVs exhibited neural protective abilities in Aß42 oligomer-treated cultures, enhancing cell viability and reducing oxidative stress. Our results demonstrate that the paracrine signaling provided by tissue context-dependent EVs derived from hiPSCs elicit distinct responses to impact the physiological state of cortical spheroids. Overall, this study advances our understanding of cell‒cell communication in the stem cell microenvironment and provides possible therapeutic options for treating neural degeneration.


Assuntos
Ectoderma/citologia , Vesículas Extracelulares/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Mesoderma/citologia , Esferoides Celulares/citologia , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Ectoderma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesoderma/metabolismo , MicroRNAs/genética , Nanopartículas , Comunicação Parácrina , Tamanho da Partícula , Esferoides Celulares/metabolismo
18.
Int J Pharm ; 566: 717-730, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31202901

RESUMO

Liposome based delivery systems provide a promising strategy for treatment of neurodegenerative diseases. A rational design of brain-targeted liposomes can support the development of more efficient treatments with drugs and gene materials. Here, we characterized surface modified liposomes with transferrin (Tf) protein and penetratin (Pen), a cell-penetrating peptide, for efficient and targeted gene delivery to brain cells. PenTf-liposomes efficiently encapsulated plasmid DNA, protected them against enzymatic degradation and exhibited a sustained in vitro release kinetics. The formulation demonstrated low cytotoxicity and was non-hemolytic. Liposomes were internalized into cells mainly through energy-dependent pathways especially clathrin-mediated endocytosis. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using PenTf-liposomes compared to unmodified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. Rationally designed bifunctional targeted-liposomes provide an efficient tool for improving the targetability and efficacy of synthesized delivery systems. This investigation of liposomal properties attempted to address cell differences, as well as, vector differences, in gene transfectability. The findings indicate that PenTf-liposomes can be a safe and non-invasive approach to transfect neuronal cells through multiple endocytosis pathways.


Assuntos
Peptídeos Penetradores de Células/administração & dosagem , DNA/administração & dosagem , Técnicas de Transferência de Genes , Nanopartículas/administração & dosagem , Neurônios/metabolismo , Transferrina/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Lipossomos , Neurônios/efeitos dos fármacos , Plasmídeos , Ratos
20.
Int J Mol Sci ; 20(6)2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934555

RESUMO

Apolipoprotein E (apoE) is linked to the risk for Alzheimer's disease (AD) and thus has been suggested to be an important therapeutic target. In our drug screening effort, we identified Ondansetron (OS), an FDA-approved 5-HT3 antagonist, as an apoE-modulating drug. OS at low micromolar concentrations significantly increased apoE secretion from immortalized astrocytes and primary astrocytes derived from apoE3 and apoE4-targeted replacement mice without generating cellular toxicity. Other 5-HT3 antagonists also had similar effects as OS, though their effects were milder and required higher concentrations. Antagonists for other 5-HT receptors did not increase apoE secretion. OS also increased mRNA and protein levels of the ATB-binding cassette protein A1 (ABCA1), which is involved in lipidation and secretion of apoE. Accordingly, OS increased high molecular weight apoE. Moreover, the liver X receptor (LXR) and ABCA1 antagonists blocked the OS-induced increase of apoE secretion, indicating that the LXR-ABCA1 pathway is involved in the OS-mediated facilitation of apoE secretion from astrocytes. The effects of OS on apoE and ABCA1 were also observed in human astrocytes derived from induced pluripotent stem cells (iPSC) carrying the APOE ε3/ε3 and APOE ε4/ε4 genotypes. Oral administration of OS at clinically-relevant doses affected apoE levels in the liver, though the effects in the brain were not observed. Collectively, though further studies are needed to probe its effects in vivo, OS could be a potential therapeutic drug for AD by modulating poE metabolism through the LXR-ABCA1 pathway.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteínas E/metabolismo , Receptores X do Fígado/metabolismo , Ondansetron/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Animais , Apolipoproteína E3/genética , Apolipoproteína E4/genética , Astrócitos/metabolismo , Células Cultivadas , Humanos , Masculino , Camundongos Transgênicos
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