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Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

ABSTRACT: Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8%?±?1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3%?±?5.1%, p?=?0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection.

SUBMITTER: Chang MF

PROVIDER: S-EPMC5066268 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

Chang Ming-Fong MF Hsieh Jung-Hsien JH Chiang Hao H Kan Hung-Wei HW Huang Cho-Min CM Chellis Luke L Lin Bo-Shiou BS Miaw Shi-Chuen SC Pan Chun-Liang CL Chao Chi-Chao CC Hsieh Sung-Tsang ST

Scientific reports 20161017

Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI ...[more]

PMID: 27748450

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Project description:Methyl-CpG-binding protein 2 (MeCP2), a protein with affinity for methylated cytosines, is crucial for neuronal development and function. MeCP2 regulates gene expression through activation, repression and chromatin remodeling. Mutations in MeCP2 cause Rett syndrome, and these patients display impaired nociception. We observed an increase in MeCP2 expression in mouse dorsal root ganglia (DRG) after peripheral nerve injury. The functional implication of increased MeCP2 is largely unknown. To identify regions of the genome bound by MeCP2 in the DRG and the changes induced by nerve injury, a chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) was performed 4 weeks after spared nerve injury (SNI).While the number of binding sites across the genome remained similar in the SNI model and sham control, SNI induced the redistribution of MeCP2 to transcriptionally relevant regions. To determine how differential binding of MeCP2 can affect gene expression in the DRG, we investigated mmu-miR-126, a microRNA locus that had enriched MeCP2 binding in the SNI model. Enriched MeCP2 binding to miR-126 locus after nerve injury repressed miR-126 expression, and this was not mediated by alterations in methylation pattern at the miR-126 locus. Downregulation of miR-126 resulted in the upregulation of its two target genes Dnmt1 and Vegfa in Neuro 2A cells and in SNI model compared to control. These target genes were significantly downregulated in Mecp2-null mice compared to wild-type littermates, indicating a regulatory role for MeCP2 in activating Dnmt1 and Vegfa expression. Intrathecal delivery of miR-126 was not sufficient to reverse nerve injury-induced mechanical and thermal hypersensitivity, but decreased Dnmt1 and Vegfa expression in the DRG.Our study shows a regulatory role for MeCP2 in that changes in global redistribution can result in direct and indirect modulation of gene expression in the DRG. Alterations in genome-wide binding of MeCP2 therefore provide a molecular basis for a better understanding of epigenetic regulation-induced molecular changes underlying nerve injury.

Project description:BackgroundATP-sensitive potassium (KATP) channels in neurons mediate neuroprotection, they regulate membrane excitability, and they control neurotransmitter release. Because loss of DRG neuronal KATP currents is involved in the pathophysiology of pain after peripheral nerve injury, we characterized the distribution of the KATP channel subunits in rat DRG, and determined their alterations by painful axotomy using RT-PCR, immunohistochemistry and electron microscopy.ResultsPCR demonstrated Kir6.1, Kir6.2, SUR1 and SUR2 transcripts in control DRG neurons. Protein expression for all but Kir6.1 was confirmed by Western blots and immunohistochemistry. Immunostaining of these subunits was identified by fluorescent and confocal microscopy in plasmalemmal and nuclear membranes, in the cytosol, along the peripheral fibers, and in satellite glial cells. Kir6.2 co-localized with SUR1 subunits. Kir6.2, SUR1, and SUR2 subunits were identified in neuronal subpopulations, categorized by positive or negative NF200 or CGRP staining. KATP current recorded in excised patches was blocked by glybenclamide, but preincubation with antibody against SUR1 abolished this blocking effect of glybenclamide, confirming that the antibody targets the SUR1 protein in the neuronal plasmalemmal membrane. In the myelinated nerve fibers we observed anti-SUR1 immunostaining in regularly spaced funneled-shaped structures. These structures were identified by electron microscopy as Schmidt-Lanterman incisures (SLI) formed by the Schwann cells. Immunostaining against SUR1 and Kir6.2 colocalized with anti-Caspr at paranodal sites.DRG excised from rats made hyperalgesic by spinal nerve ligation exhibited similar staining against Kir6.2, SUR1 or SUR2 as DRG from controls, but showed decreased prevalence of SUR1 immunofluorescent NF200 positive neurons. In DRG and dorsal roots proximal to axotomy SLI were smaller and showed decreased SUR1 immunofluorescence.ConclusionsWe identified Kir6.2/SUR1 and Kir6.2/SUR2 KATP channels in rat DRG neuronal somata, peripheral nerve fibers, and glial satellite and Schwann cells, in both normal state and after painful nerve injury. This is the first report of KATP channels in paranodal sites adjacent to nodes of Ranvier and in the SLI of the Schwann cells. After painful axotomy KATP channels are downregulated in large, myelinated somata and also in SLI, which are also of smaller size compared to controls.Because KATP channels may have diverse functional roles in neurons and glia, further studies are needed to explore the potential of KATP channels as targets of therapies against neuropathic pain and neurodegeneration.

Dataset Information

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

Publications

Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection.

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