RESUMO
In Parkinson's disease and other synucleinopathies, fibrillar forms of α-synuclein (aSyn) are hypothesized to structurally convert and pathologize endogenous aSyn, which then propagates through the neural connections, forming Lewy pathologies and ultimately causing neurodegeneration. Inoculation of mouse-derived aSyn preformed fibrils (PFFs) into the unilateral striatum of wild-type mice causes widespread aSyn pathologies in the brain through the neural network. Here, we used the local injection of antisense oligonucleotides (ASOs) against Snca mRNA to confine the area of endogenous aSyn protein reduction and not to affect the PFFs properties in this model. We then varied the timing and location of ASOs injection to examine their impact on the initiation and propagation of aSyn pathologies in the whole brain and the therapeutic effect using abnormally-phosphorylated aSyn (pSyn) as an indicator. By injecting ASOs before or 0-14 days after the PFFs were inoculated into the same site in the left striatum, the reduction in endogenous aSyn in the striatum leads to the prevention and inhibition of the regional spread of pSyn pathologies to the whole brain including the contralateral right hemisphere. ASO post-injection inhibited extension from neuritic pathologies to somatic ones. Moreover, injection of ASOs into the right striatum prevented the remote regional spread of pSyn pathologies from the left striatum where PFFs were inoculated and no ASO treatment was conducted. This indicated that the reduction in endogenous aSyn protein levels at the propagation destination site can attenuate pSyn pathologies, even if those at the propagation initiation site are not inhibited, which is consistent with the original concept of prion-like propagation that endogenous aSyn is indispensable for this regional spread. Our results demonstrate the importance of recruiting endogenous aSyn in this neural network propagation model and indicate a possible potential for ASO treatment in synucleinopathies.
Assuntos
Camundongos Endogâmicos C57BL , Rede Nervosa , Oligonucleotídeos Antissenso , alfa-Sinucleína , Animais , Camundongos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Corpo Estriado/efeitos dos fármacos , Modelos Animais de Doenças , Rede Nervosa/metabolismo , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/patologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/administração & dosagem , RNA Mensageiro/metabolismoRESUMO
Neuropathic pain, a heterogeneous condition, affects 7%-10% of the general population. To date, efficacious and safe therapeutic approaches remain limited. Antisense oligonucleotide (ASO) therapy has opened the door to treat spinal muscular atrophy, with many ongoing clinical studies determining its therapeutic utility. ASO therapy for neuropathic pain and peripheral nerve disease requires efficient gene delivery and knockdown in both the dorsal root ganglion (DRG) and sciatic nerve, key tissues for pain signaling. We previously developed a new DNA/RNA heteroduplex oligonucleotide (HDO) technology that achieves highly efficient gene knockdown in the liver. Here, we demonstrated that intravenous injection of HDO, comprising an ASO and its complementary RNA conjugated to α-tocopherol, silences endogenous gene expression more than 2-fold in the DRG, and sciatic nerve with higher potency, efficacy, and broader distribution than ASO alone. Of note, we observed drastic target suppression in all sizes of neuronal DRG populations by in situ hybridization. Our findings establish HDO delivery as an investigative and potentially therapeutic platform for neuropathic pain and peripheral nerve disease.
RESUMO
Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.
Assuntos
Barreira Hematoencefálica , RNA , Animais , Sistema Nervoso Central/metabolismo , Colesterol/metabolismo , DNA/metabolismo , Camundongos , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , RNA/metabolismo , Ratos , RoedoresRESUMO
BACKGROUND: The vesicular GABA transporter (VGAT) loads GABA and glycine from the neuronal cytoplasm into synaptic vesicles. To address functional importance of VGAT during embryonic development, we generated global VGAT knockout mice and analyzed them. RESULTS: VGAT knockouts at embryonic day (E) 18.5 exhibited substantial increases in overall GABA and glycine, but not glutamate, contents in the forebrain. Electrophysiological recordings from E17.5-18.5 spinal cord motoneurons demonstrated that VGAT knockouts presented no spontaneous inhibitory postsynaptic currents mediated by GABA and glycine. Histological examination of E18.5 knockout fetuses revealed reductions in the trapezius muscle, hepatic congestion and little alveolar spaces in the lung, indicating that the development of skeletal muscle, liver and lung in these mice was severely affected. CONCLUSION: VGAT is fundamental for the GABA- and/or glycine-mediated transmission that supports embryonic development. VGAT knockout mice will be useful for further investigating the roles of VGAT in normal physiology and pathophysiologic processes.
Assuntos
Desenvolvimento Embrionário , Camundongos Knockout , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Animais , Fissura Palatina/genética , Feminino , Genótipo , Glutamato Descarboxilase/genética , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Hérnia Umbilical/genética , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Pulmão/citologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Técnicas de Patch-Clamp , Gravidez , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismoRESUMO
Neurons expressing the calcium-binding protein parvalbumin (PV) constitute an abundant subpopulation of GABAergic neurons in the cerebral cortex. However, PV is not unique to the GABAergic neurons of the forebrain, but is also expressed in a small number of pyramidal neurons and in a large number of thalamic neurons. In order to summarize the PV neurons in the forebrain, we employed the PV-Cre transgenic mice in the present study. In the progeny of crossbreed between PV-Cre mice and GFP-Cre reporter mice, we found that the GFP-positive neurons include many excitatory neurons in the neocortex and the thalamus as well as GABAergic neurons in the cerebral cortex and basal ganglia. All the reported PV-positive GABAergic neurons in the cerebral cortex and the basal ganglia seemed to be included in the GFP-positive cells. We found GFP-positive layer V pyramidal neurons inhabit a broader neocortical area than was previously reported. They were located in the primary somatosensory, motor, and visual areas. The somatosensory area of the neocortex contained the greatest number of PV-positive pyramidal neurons. A large number of thalamic relay neurons and virtually all the reticular thalamic neurons appeared as GFP-positive. Thalamic relay nucleus and a neocortical area for the same modality corresponded and seemed to contain a characteristic amount of PV-positive excitatory neurons.
Assuntos
Integrases/genética , Neurônios/metabolismo , Parvalbuminas/genética , Parvalbuminas/metabolismo , Prosencéfalo/citologia , Adenoviridae/genética , Animais , Calbindina 2 , Calbindinas , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismo , Tálamo/citologia , Distribuição Tecidual , Ácido gama-Aminobutírico/metabolismoRESUMO
Although neocortical GABAergic (gamma-aminobutyric acidergic) interneurons have been the focus of intense study, especially in the rat, a consensus view of the functional diversity and organization of inhibitory cortical neurons has not yet been achieved. To better analyze GABAergic neurons in the rat, we used a bacterial artificial chromosome (BAC) construct and established 2 lines of transgenic rats that coexpress Venus, a yellow fluorescent protein, with the vesicular GABA transporter. The brain GABA content from both transgenic lines was similar to the level found in wild-type rats. In the frontal cortex, Venus was expressed in >95% of GABAergic neurons, most of which also expressed at least one of 6 biochemical markers, including alpha-actitin-2, which preferentially labeled late-spiking neurogliaform cells. Taking advantage of the fact that Venus expression allows for targeted recording from all classes of nonpyramidal cells, irrespective of their somatic morphologies, we demonstrated that fast-spiking neurons, which were heterogeneous in somatic size as well as vertical dendritic projection, had relatively uniform horizontal dimensions, suggesting a cell type-specific columnar input territory. Our data demonstrate the benefits of VGAT-Venus rats for investigating GABAergic circuits, as well as the feasibility of using BAC technology in rats to label subsets of specific, genetically defined neurons.
Assuntos
Proteínas de Bactérias , Mapeamento Encefálico/métodos , Córtex Cerebral/metabolismo , Interneurônios/metabolismo , Proteínas Luminescentes , Inibição Neural/fisiologia , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Geneticamente Modificados , Microscopia de Fluorescência/métodos , Ratos/genética , Distribuição TecidualRESUMO
Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator. It has been reported that MBF1 changed its subcellular localization from cytoplasm into nuclei with a transcriptional activator for activation of a target gene expression in animals. We found that Arabidopsis MBF1s (AtMBF1s) predominantly localize in nucleolus. We previously reported that plant MBF1s were rapidly induced by several stresses, whereas animal MBF1s were not induced. Therefore, we suggest that MBF1-function in plants is controlled on the level of transcriptional induction but not by nuclear translocation, dissimilar from the case of MBF1s from animals.
Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/química , Nucléolo Celular/química , Transativadores/análise , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucléolo Celular/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Imunoquímica , Transativadores/genética , Transativadores/metabolismoRESUMO
The type-2 glycine transporter GLYT2 is expressed in glycinergic neurons and is involved in the termination of inhibitory neurotransmission at strychnine-sensitive glycinergic synapses. We isolated cDNA of a GLYT2 isoform, GLYT2a, from mouse brain, and found that it contains a coding sequence of 798 amino acids. We also isolated and characterized the mouse GLYT2 (mGLYT2) gene, which was found to be divided into 18 exons and spread over 55 kb. 5'-rapid amplification of cDNA ends analyses demonstrated the existence of another two isoforms, mGLYT2b and mGLYT2c, in addition to mGLYT2a. Both mGLYT2b and mGLYT2c would produce a protein eight amino acids shorter than mGLYT2a. Analysis of the genomic clones encompassing the 5'-exons revealed that the three transcripts arose from a single gene by alternative splicing. RT-PCR analysis indicated that all three mGLYT2 isoforms were expressed at high levels in brain stem and spinal cord. These data will be useful for investigating the function of GLYT2 proteins and glycinergic neurons by gene targeting experiments.
Assuntos
Processamento Alternativo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Sequência de Bases , DNA Complementar , Proteínas da Membrana Plasmática de Transporte de Glicina , Humanos , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Neuronal expression of the mouse glutamate decarboxylase 67 (mGAD67) gene occurs exclusively in neurons that synthesize and release GABA (GABAergic neurons). This gene is also expressed in pancreatic islet cells and testicular spermatocytes. In order to elucidate the molecular mechanisms underlying the regulation of mGAD67 gene expression, we isolated and characterized the 5'-flanking region of this gene. Sequence analysis of a 10.2-kb DNA fragment of this gene containing a promoter region (8.4 kb) and noncoding exons 0A and 0B revealed the presence of numerous potential neuron-specific cis-regulatory elements. Functional analysis of the 5'-flanking region of exons 0A and 0B by transient transfection into cultured cells revealed that the region -98 to -52 close to exon 0A is important for the transcriptional activity of both exons 0A and 0B. In addition, we used transgenic mice to examine the expression pattern conferred by the 10.2 kb DNA fragment of the mGAD67 gene fused to the bacterial lacZ reporter gene. Transgene expression was observed in neurons of particular brain regions containing abundant GABAergic neurons such as the basal ganglia, in pancreatic islet cells and in testicular spermatocytes and spermatogonia. These results suggest that the 10.2 kb DNA fragment of the mGAD67 gene contains regulatory elements essential for its targeted expression in GABAergic neurons, islet cells and spermatocytes.
Assuntos
Glutamato Descarboxilase/genética , Regiões Promotoras Genéticas/genética , Região 5'-Flanqueadora , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Genes Reporter , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de ÓrgãosRESUMO
The vesicular GABA transporter (VGAT) loads GABA from neuronal cytoplasm into synaptic vesicles and is selectively expressed in inhibitory neurons that contain GABA and/or glycine. To elucidate the molecular mechanisms of mouse VGAT (mVGAT) gene expression, we have isolated and characterized the mVGAT gene. The mVGAT gene was found to be 4.7 kilobases in size and to contain three exons and two introns by comparison of the cloned genomic DNA with the cDNA (termed mVGATa) sequence reported by Sagne et al. [FEBS Lett. 417 (1997) 177]. Analysis of transcripts and genomic DNA revealed an alternatively spliced mVGAT isoform (termed mVGATb) that retains intron 2 of mVGATa as an exon. This alternative transcript specifies 514 amino acid residues identical to VGATa followed by a unique C-terminal sequence of 11 amino acids encoded by intron 2. Fluorescent in situ hybridization studies showed that the mVGAT gene is localized on chromosome 2. One major transcription start site of the mVGAT gene is an A residue 209 bp upstream from the translational initiation site, as shown using the 5'-RACE method. RT-PCR analysis revealed that the mVGAT gene was expressed at a high level in retinoic acid-treated P19 embryonal carcinoma cells, at a very low level in non-treated P19 cells, and not detectably expressed in Neuro-2a neuroblastoma cells. Sequence analysis of the 5'-flanking region revealed a number of putative regulatory elements including Sp1, Egr-1 and Pitx binding sites. In transient transfection assays, 2 kilobases of the mVGAT 5'-flanking region generated similar levels of luciferase reporter activity in three kinds of cultured cells. Deletion analysis and gel mobility shift assays demonstrated that the region -161 to +155 contained the basal promoter activity of the mVGAT gene and that an activating region from -49 to -27 bound an Sp1-like protein. These results suggest a possible mechanism for regulation of the expression of the mVGAT gene.