RESUMO
Divalent short-interfering RNA (siRNA) holds promise as a therapeutic approach allowing for the sequence-specific modulation of a target gene within the central nervous system (CNS). However, an siRNA modality capable of simultaneously modulating gene pairs would be invaluable for treating complex neurodegenerative disorders, where more than one pathway contributes to pathogenesis. Currently, the parameters and scaffold considerations for multi-targeting nucleic acid modalities in the CNS are undefined. Here, we propose a framework for designing unimolecular 'dual-targeting' divalent siRNAs capable of co-silencing two genes in the CNS. We systematically adjusted the original CNS-active divalent siRNA and identified that connecting two sense strands 3' and 5' through an intra-strand linker enabled a functional dual-targeting scaffold, greatly simplifying the synthetic process. Our findings demonstrate that the dual-targeting siRNA supports at least two months of maximal distribution and target silencing in the mouse CNS. The dual-targeting divalent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g. Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting divalent siRNAs against each gene. This work enhances the potential for CNS modulation of disease-related gene pairs using a unimolecular siRNA.
Assuntos
Sistema Nervoso Central , RNA Interferente Pequeno , Animais , Humanos , Camundongos , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Apolipoproteínas E/genética , Sistema Nervoso Central/metabolismo , Inativação Gênica , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/terapia , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/químicaRESUMO
A missense mutation in the transcription repressor Nucleus accumbens-associated 1 (NACC1) gene at c.892C>T (p.Arg298Trp) on chromosome 19 causes severe neurodevelopmental delay ( Schoch et al., 2017). To model this disorder, we engineered the first mouse model with the homologous mutation (Nacc1+/R284W ) and examined mice from E17.5 to 8â months. Both genders had delayed weight gain, epileptiform discharges and altered power spectral distribution in cortical electroencephalogram, behavioral seizures, and marked hindlimb clasping; females displayed thigmotaxis in an open field. In the cortex, NACC1 long isoform, which harbors the mutation, increased from 3 to 6â months, whereas the short isoform, which is not present in humans and lacks aaR284 in mice, rose steadily from postnatal day (P) 7. Nuclear NACC1 immunoreactivity increased in cortical pyramidal neurons and parvalbumin containing interneurons but not in nuclei of astrocytes or oligodendroglia. Glial fibrillary acidic protein staining in astrocytic processes was diminished. RNA-seq of P14 mutant mice cortex revealed over 1,000 differentially expressed genes (DEGs). Glial transcripts were downregulated and synaptic genes upregulated. Top gene ontology terms from upregulated DEGs relate to postsynapse and ion channel function, while downregulated DEGs enriched for terms relating to metabolic function, mitochondria, and ribosomes. Levels of synaptic proteins were changed, but number and length of synaptic contacts were unaltered at 3â months. Homozygosity worsened some phenotypes including postnatal survival, weight gain delay, and increase in nuclear NACC1. This mouse model simulates a rare form of autism and will be indispensable for assessing pathophysiology and targets for therapeutic intervention.
Assuntos
Transtorno Autístico , Fatores de Transcrição , Animais , Feminino , Humanos , Masculino , Camundongos , Mutação/genética , Proteínas de Neoplasias/genética , Isoformas de Proteínas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Aumento de PesoRESUMO
Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.
Assuntos
Doença de Huntington , Proteína 3 Homóloga a MutS , Expansão das Repetições de Trinucleotídeos , Animais , Camundongos , Corpo Estriado/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/terapia , Doença de Huntington/metabolismo , Neostriado/metabolismo , RNA de Cadeia Dupla , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Proteína 3 Homóloga a MutS/genéticaRESUMO
Expansion of a triplet repeat tract in exon 1 of the HTT gene causes Huntington's disease (HD). The mutant HTT protein (mHTT) has numerous aberrant interactions with diverse, pleiomorphic effects. Lowering mHTT is a promising approach to treat HD, but it is unclear when lowering should be initiated, how much is necessary, and what duration should occur to achieve benefits. Furthermore, the effects of mHTT lowering on brain lipids have not been assessed. Using a mHtt-inducible mouse model, we analyzed mHtt lowering initiated at different ages and sustained for different time-periods. mHTT protein in cytoplasmic and synaptic compartments of the striatum was reduced 38-52%; however, there was minimal lowering of mHTT in nuclear and perinuclear regions where aggregates formed at 12 months of age. Total striatal lipids were reduced in 9-month-old LacQ140 mice and preserved by mHtt lowering. Subclasses important for white matter structure and function including ceramide (Cer), sphingomyelin (SM), and monogalactosyldiacylglycerol (MGDG), contributed to the reduction in total lipids. Phosphatidylinositol (PI), phosphatidylserine (PS), and bismethyl phosphatidic acid (BisMePA) were also changed in LacQ140 mice. Levels of all subclasses except ceramide were preserved by mHtt lowering. mRNA expression profiling indicated that a transcriptional mechanism contributes to changes in myelin lipids, and some but not all changes can be prevented by mHtt lowering. Our findings suggest that early and sustained reduction in mHtt can prevent changes in levels of select striatal proteins and most lipids, but a misfolded, degradation-resistant form of mHTT hampers some benefits in the long term.
Assuntos
Doença de Huntington , Substância Branca , Camundongos , Animais , Substância Branca/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Corpo Estriado/metabolismo , Proteínas Mutantes/genética , Ceramidas/metabolismo , Lipídeos , Modelos Animais de DoençasRESUMO
Oligonucleotides is an emerging class of chemically-distinct therapeutic modalities, where extensive chemical modifications are fundamental for their clinical applications. Inter-nucleotide backbones are critical to the behaviour of therapeutic oligonucleotides, but clinically explored backbone analogues are, effectively, limited to phosphorothioates. Here, we describe the synthesis and bio-functional characterization of an internucleotide (E)-vinylphosphonate (iE-VP) backbone, where bridging oxygen is substituted with carbon in a locked stereo-conformation. After optimizing synthetic pathways for iE-VP-linked dimer phosphoramidites in different sugar contexts, we systematically evaluated the impact of the iE-VP backbone on oligonucleotide interactions with a variety of cellular proteins. Furthermore, we systematically evaluated the impact of iE-VP on RNA-Induced Silencing Complex (RISC) activity, where backbone stereo-constraining has profound position-specific effects. Using Huntingtin (HTT) gene causative of Huntington's disease as an example, iE-VP at position 6 significantly enhanced the single mismatch discrimination ability of the RISC without negative impact on silencing of targeting wild type htt gene. These findings suggest that the iE-VP backbone can be used to modulate the activity and specificity of RISC. Our study provides (i) a new chemical tool to alter oligonucleotide-enzyme interactions and metabolic stability, (ii) insight into RISC dynamics and (iii) a new strategy for highly selective SNP-discriminating siRNAs.
Assuntos
Doença de Huntington/genética , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Alelos , Humanos , OrganofosfonatosRESUMO
X-Linked Dystonia-Parkinsonism (XDP) is a neurodegenerative disease affecting individuals with ancestry to the island of Panay in the Philippines. In recent years there has been considerable progress at elucidating the genetic basis of XDP and candidate disease mechanisms in patient-derived cellular models, but the neural substrates that give rise to XDP in vivo are still poorly understood. Previous studies of limited XDP postmortem brain samples have reported a selective dropout of medium spiny neurons within the striatum, although neuroimaging of XDP patients has detected additional abnormalities in multiple brain regions beyond the basal ganglia. Given the need to fully define the CNS structures that are affected in this disease, we created a brain bank in Panay to serve as a tissue resource for detailed studies of XDP-related neuropathology. Here we describe this platform, from donor recruitment and consent to tissue collection, processing, and storage, that was assembled within a predominantly rural region of the Philippines with limited access to medical and laboratory facilities. Thirty-six brains from XDP individuals have been collected over an initial 4 years period. Tissue quality was assessed based on histologic staining of cortex, RNA integrity scores, detection of neuronal transcripts in situ by fluorescent hybridization chain reaction, and western blotting of neuronal and glial proteins. The results indicate that this pipeline preserves tissue integrity to an extent compatible with a range of morphologic, molecular, and biochemical analyses. Thus the algorithms that we developed for working in rural communities may serve as a guide for establishing similar brain banks for other rare diseases in indigenous populations.
Assuntos
Distonia , Distúrbios Distônicos , Doenças Neurodegenerativas , Encéfalo/diagnóstico por imagem , Distúrbios Distônicos/genética , Doenças Genéticas Ligadas ao Cromossomo X , HumanosRESUMO
Molecular changes at synapses are thought to underly the deficits in motor and cognitive dysfunction seen in Huntington's disease (HD). Previously we showed in synaptosome preparations age dependent changes in levels of selected proteins examined by western blot assay in the striatum of Q140/Q140 HD mice. To assess if CAG repeat length influenced protein changes at the synapse, we examined synaptosomes from 6-month old heterozygote HD mice with CAG repeat lengths ranging from 50 to 175. Analysis of 19 selected proteins showed that increasing CAG repeat length in huntingtin (HTT) increased the number of affected proteins in HD striatal synaptosomes. Moreover, SDS-soluble total HTT (WT plus mutant HTT) and pThr3 HTT were reduced with increasing CAG repeat length, and there was no pSer421 mutant HTT detected in any HD mice. A LC-MS/MS and bioinfomatics study of synaptosomes from 2 and 6-month old striatum and cortex of Q140/Q7 HD mice showed enrichment of synaptic proteins and an influence of age, gender and brain region on the number of protein changes. HD striatum at 6 months had the most protein changes that included many HTT protein interactors, followed by 2-month old HD striatum, 2-month old HD cortex and 6-month HD cortex. SDS-insoluble mutant HTT was detected in HD striatal synaptosomes consistent with the presence of aggregates. Proteins changed in cortex differed from those in striatum. Pathways affected in HD striatal synaptosomes that were not identified in whole striatal lysates of the same HD mouse model included axon guidance, focal adhesion, neurotrophin signaling, regulation of actin cytoskeleton, endocytosis, and synaptic vesicle cycle. Results suggest that synaptosomes prepared from HD mice are highly informative for monitoring protein changes at the synapse and may be preferred for assessing the effects of experimental therapies on synaptic function in HD.
Assuntos
Encéfalo/metabolismo , Doença de Huntington/metabolismo , Sinapses/metabolismo , Fatores Etários , Animais , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Proteína Huntingtina/genética , Masculino , Camundongos Endogâmicos C57BL , Neostriado/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Fosforilação , Sinapses/ultraestruturaRESUMO
The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology.
Assuntos
Doença de Huntington/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Adulto , Idoso , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Doença de Huntington/genética , Proteína 1 Associada a ECH Semelhante a Kelch/química , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/química , Células-Tronco Neurais/metabolismo , Fármacos Neuroprotetores/farmacologia , Conformação Proteica/efeitos dos fármacos , Ratos , Transdução de SinaisRESUMO
Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at â¼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles.
Assuntos
Colesterol/química , Vesículas Extracelulares/química , Proteína Huntingtina/genética , RNA Interferente Pequeno/química , Animais , Células Cultivadas , Humanos , Camundongos , Mutação , Propilenoglicóis/químicaRESUMO
Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. However, scalable methods to isolate and manufacture exosomes from mesenchymal stem cells are lacking, a limitation to the clinical translation of exosome technology. We evaluate mesenchymal stem cells from different sources and find that umbilical cord-derived mesenchymal stem cells produce the highest exosome yield. To optimize exosome production, we cultivate umbilical cord-derived mesenchymal stem cells in scalable microcarrier-based three-dimensional (3D) cultures. In combination with the conventional differential ultracentrifugation, 3D culture yields 20-fold more exosomes (3D-UC-exosomes) than two-dimensional cultures (2D-UC-exosomes). Tangential flow filtration (TFF) in combination with 3D mesenchymal stem cell cultures further improves the yield of exosomes (3D-TFF-exosomes) 7-fold over 3D-UC-exosomes. 3D-TFF-exosomes are seven times more potent in small interfering RNA (siRNA) transfer to neurons compared with 2D-UC-exosomes. Microcarrier-based 3D culture and TFF allow scalable production of biologically active exosomes from mesenchymal stem cells. These findings lift a major roadblock for the clinical utility of mesenchymal stem cell exosomes.
Assuntos
Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Feminino , Inativação Gênica , Células-Tronco Mesenquimais/citologia , Camundongos , Neurônios/metabolismo , Proteoma , RNA Interferente Pequeno/genética , Esferoides Celulares , Cordão Umbilical/citologiaRESUMO
Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Proteína Huntingtina/genética , Neurônios/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Barreira Hematoencefálica/fisiopatologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Artérias Carótidas/fisiologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/química , Inativação Gênica , Terapia Genética/métodos , Humanos , Proteína Huntingtina/antagonistas & inibidores , Interações Hidrofóbicas e Hidrofílicas , Manitol/administração & dosagem , Neurônios/patologia , Fosforilcolina/administração & dosagem , Fosforilcolina/química , RNA Interferente Pequeno/química , RatosRESUMO
Ligand-conjugated siRNAs have the potential to achieve targeted delivery and efficient silencing in neurons following local administration in the central nervous system (CNS). We recently described the activity and safety profile of a docosahexaenoic acid (DHA)-conjugated, hydrophobic siRNA (DHA-hsiRNA) targeting Huntingtin (Htt) mRNA in mouse brain. Here, we report the synthesis of an amide-modified, phosphocholine-containing DHA-hsiRNA conjugate (PC-DHA-hsiRNA), which closely resembles the endogenously esterified biological structure of DHA. We hypothesized that this modification may enhance neuronal delivery in vivo. We demonstrate that PC-DHA-hsiRNA silences Htt in mouse primary cortical neurons and astrocytes. After intrastriatal delivery, Htt-targeting PC-DHA-hsiRNA induces â¼80% mRNA silencing and 71% protein silencing after 1 week. However, PC-DHA-hsiRNA did not substantially outperform DHA-hsiRNA under the conditions tested. Moreover, at the highest locally administered dose (4 nmol, 50 µg), we observe evidence of PC-DHA-hsiRNA-mediated reactive astrogliosis. Lipophilic ligand conjugation enables siRNA delivery to neural tissues, but rational design of functional, nontoxic siRNA conjugates for CNS delivery remains challenging.
Assuntos
Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Tecido Parenquimatoso/metabolismo , RNA Interferente Pequeno/síntese química , Animais , Encéfalo/patologia , Ácidos Docosa-Hexaenoicos/química , Estabilidade de Medicamentos , Inativação Gênica , Proteína Huntingtina/genética , Camundongos , Fosforilcolina/química , Interferência de RNA , RNA Mensageiro , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Serina/química , Resultado do TratamentoRESUMO
Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.
Assuntos
Proteínas do Capsídeo/metabolismo , Sistema Nervoso Central/metabolismo , Peptídeos/genética , Transdução Genética , Animais , Células CHO , Proteínas do Capsídeo/genética , Gatos , Linhagem Celular , Cricetulus , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/administração & dosagem , Proteína Huntingtina/antagonistas & inibidores , Proteína Huntingtina/genética , Camundongos , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntington's disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntington's disease and other neurodegenerative disorders.
Assuntos
Exossomos/genética , Proteína Huntingtina/genética , Neurônios/metabolismo , RNA Interferente Pequeno/administração & dosagem , Animais , Células Cultivadas , Regulação da Expressão Gênica , Inativação Gênica , Terapia Genética , Humanos , Proteína Huntingtina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologiaRESUMO
Sphingosine kinase 2 (SPK2) and autophagy are both involved in brain preconditioning, but whether preconditioning-induced SPK2 up-regulation and autophagy activation are linked mechanistically remains to be elucidated. In this study, we used in vitro and in vivo models to explore the role of SPK2-mediated autophagy in isoflurane and hypoxic preconditioning. In primary mouse cortical neurons, both isoflurane and hypoxic preconditioning induced autophagy. Isoflurane and hypoxic preconditioning protected against subsequent oxygen glucose deprivation or glutamate injury, whereas pretreatment with autophagy inhibitors (3-methyladenine or KU55933) abolished preconditioning-induced tolerance. Pretreatment with SPK2 inhibitors (ABC294640 and SKI-II) or SPK2 knockdown prevented preconditioning-induced autophagy. Isoflurane also induced autophagy in mouse in vivo as shown by Western blots for LC3 and p62, LC3 immunostaining, and electron microscopy. Isoflurane-induced autophagy in mice lacking the SPK1 isoform (SPK1(-/-)), but not in SPK2(-/-)mice. Sphingosine 1-phosphate and the sphingosine 1-phosphate receptor agonist FTY720 did not protect against oxygen glucose deprivation in cultured neurons and did not alter the expression of LC3 and p62, suggesting that SPK2-mediated autophagy and protections are not S1P-dependent. Beclin 1 knockdown abolished preconditioning-induced autophagy, and SPK2 inhibitors abolished isoflurane-induced disruption of the Beclin 1/Bcl-2 association. These results strongly indicate that autophagy is involved in isoflurane preconditioning both in vivo and in vitro and that SPK2 contributes to preconditioning-induced autophagy, possibly by disrupting the Beclin 1/Bcl-2 interaction.
Assuntos
Autofagia , Córtex Cerebral/metabolismo , Precondicionamento Isquêmico , Neurônios/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Anestésicos Inalatórios/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Células Cultivadas , Córtex Cerebral/citologia , Inibidores Enzimáticos/farmacologia , Isoflurano/farmacologia , Lactosilceramidas/genética , Lactosilceramidas/metabolismo , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Neurônios/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Piridinas/farmacologia , Pironas/farmacologia , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
A mutation in the huntingtin (Htt) gene produces mutant Htt and Huntington's disease (HD), a neurodegenerative disorder. HD patients have oxidative damage in the brain, but the causes are unclear. Compared with controls, we found brain levels of NADPH oxidase (NOX) activity, which produces reactive oxygen species (ROS), elevated in human HD postmortem cortex and striatum and highest in striatum of presymptomatic individuals. Synaptosome fractions from cortex and striatum of HD(140Q/140Q) mice had elevated NOX activity at 3 months of age and a further rise at 6 and 12 months compared with synaptosomes of age-matched wild-type (WT) mice. High NOX activity in primary cortical and striatal neurons of HD(140Q/140Q) mice correlated with more ROS and neurite swellings. These features and neuronal cell death were markedly reduced by treatment with NOX inhibitors such as diphenyleneiodonium (DPI), apocynin (APO) and VAS2870. The rise in ROS levels in mitochondria of HD(140Q/140Q) neurons followed the rise in NOX activity and inhibiting only mitochondrial ROS was not neuroprotective. Mutant Htt colocalized at plasma membrane lipid rafts with gp91-phox, a catalytic subunit for the NOX2 isoform. Assembly of NOX2 components at lipid rafts requires activation of Rac1 which was also elevated in HD(140Q/140Q) neurons. HD(140Q/140Q) mice bred to gp91-phox knock-out mice had lower NOX activity in the brain and in primary neurons, and neurons had normal ROS levels and significantly improved survival. These findings suggest that increased NOX2 activity at lipid rafts is an early and major source of oxidative stress and cell death in HD(140Q/140Q) neurons.
Assuntos
Doença de Huntington/enzimologia , Doença de Huntington/fisiopatologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo , Animais , Morte Celular , Modelos Animais de Doenças , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NADPH Oxidase 2 , NADPH Oxidases/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Loss-of-function mutations of the gene encoding the trafficking protein particle complex subunit 9 (Trappc9) cause autosomal recessive intellectual disability and obesity by unknown mechanisms. Genome-wide analysis links Trappc9 to nonalcoholic fatty liver disease (NAFLD). Trappc9-deficient mice have been shown to appear overweight shortly after weaning. Here, we analyzed serum biochemistry and histology of adipose and liver tissues to determine the incidence of obesity and NAFLD in Trappc9-deficient mice and combined transcriptomic and proteomic analyses, pharmacological studies, and biochemical and histological examinations of postmortem mouse brains to unveil mechanisms involved. We found that Trappc9-deficient mice presented with systemic glucose homeostatic disturbance, obesity, and NAFLD, which were relieved upon chronic treatment combining dopamine receptor D2 (DRD2) agonist quinpirole and DRD1 antagonist SCH23390. Blood glucose homeostasis in Trappc9-deficient mice was restored upon administering quinpirole alone. RNA-sequencing analysis of DRD2-containing neurons and proteomic study of brain synaptosomes revealed signs of impaired neurotransmitter secretion in Trappc9-deficient mice. Biochemical and histological studies of mouse brains showed that Trappc9-deficient mice synthesized dopamine normally, but their dopamine-secreting neurons had a lower abundance of structures for releasing dopamine in the striatum. Our study suggests that Trappc9 loss of function causes obesity and NAFLD by constraining dopamine synapse formation.
Assuntos
Dopamina , Camundongos Knockout , Animais , Camundongos , Dopamina/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Obesidade/metabolismo , Obesidade/genética , Encéfalo/metabolismo , Encéfalo/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Masculino , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/genética , Modelos Animais de Doenças , Transmissão Sináptica/efeitos dos fármacosRESUMO
Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography (EEG) and electromyography (EMG) in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester (PO)/phosphorothioate (PS) bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establishes the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.