Delayed Intrathecal Delivery of RhoA siRNA to the Contused Spinal Cord Inhibits Allodynia, Preserves White Matter, and Increases Serotonergic Fiber Growth

Otsuka, Seiji; Adamson, Crista L.; Venkatachalam, Sankar; Gibbs, Kurt M.; Kane‐Goldsmith, Noriko; Ayer, Jennie; Babiarz, Joanne; Kalinski, Hagar; Ashush, Hagit; Alpert, Evgenia; Lahav, Ron; Feinstein, Elena; Grumet, Martin

doi:10.1089/neu.2010.1568

Cited by 31 publications

(27 citation statements)

References 54 publications

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“…Comparing the amount and length of regenerating axons of our present study to these previous results, the effects of specific RhoA-downregulation are clearly less pronounced than PTEN and mTOR modulation or C3-treatment and additional lens injury. In line with our results in the optic nerve, RhoA-siRNA applied by intraspinal injections or lumbar puncture had only minor positive effects on motorical outcome in a rat spinal cord injury model although improved sensory function and white matter preservation were reported (Otsuka et al, 2011). …”

Section: Discussionsupporting

confidence: 90%

Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

Koch¹,

Tönges²,

Michel³

et al. 2014

Front. Cell. Neurosci.

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The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush.

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Section: Discussionsupporting

confidence: 90%

Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

Koch¹,

Tönges²,

Michel³

et al. 2014

Front. Cell. Neurosci.

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“…A wide range of cationic lipids and polymers, including PEI, polyamidoamine (PAMAM) dendrimers, and poly (β-amino esters) have been evaluated for nonviral delivery of pDNA and small RNA therapeutics [48-50]. Currently, only a few studies have investigated non-viral delivery of siRNA in spinal cord injury models [51-53], non-viral delivery of siRNA in spinal cord injury models [51-53]. Previously, we have reported the synthesis of PgP, a micelle forming block copolymer designed for combinatorial drug/nucleic acid delivery and demonstrated its capability for in vitro transfection of siRNA and pDNA in the presence of serum and pDNA in the uninjured rat spinal cord [39].…”

Section: Discussionmentioning

confidence: 99%

“…To our knowledge, this is the first study to show that polyplexes exhibit increased residence time after local injection into spinal cord lesion site relative to naked siRNA. Several studies have used intrathecal injection and Otsuku et al reported preferential uptake of chemically-modified siRNA at the injury site, presumably due to increased penetration in injured tissue [51] [55]. In the future, we will investigate the delivery of PgP/siRhoA polyplexes to the injured spinal cord by the intrathecal route.…”

Section: Discussionmentioning

confidence: 99%

RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model

et al. 2017

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Spinal cord injury (SCI) results in permanent loss of motor and sensory function due to developmentally-related and injured-induced changes in the extrinsic microenvironment and intrinsic neuronal biochemistry that limit plasticity and axonal regeneration. Our long term goal is to develop cationic, amphiphilic copolymers (poly (lactide-co-glycolide)-g-polyethylenimine, PgP) for combinatorial delivery of therapeutic nucleic acids (TNAs) and drugs targeting these different barriers. In this study, we evaluated the ability of PgP to deliver siRNA targeting RhoA, a critical signaling pathway activated by multiple extracellular inhibitors of axonal regeneration. After generation of rat compression SCI model, PgP/siRhoA polyplexes were locally injected into the lesion site. Relative to untreated injury only, PgP/siRhoA polyplexes significantly reduced RhoA mRNA and protein expression for up to 4 weeks post-injury. Histological analysis at 4 weeks post-injury showed that RhoA knockdown was accompanied by reduced apoptosis, cavity size, and astrogliosis and increased axonal regeneration within the lesion site. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siRhoA to the injured spinal cord and may be a promising platform for the development of combinatorial TNA/drug therapy.

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“…The lentiviral construct for RhoA silencing (lenti-shRhoA) was derived from the siRNA sequence 5′-GCCACUUAAUGUAUGUUAC-3′ (Otsuka et al, 2011) by using the pLVTHM vector. A scrambled control shRNA (lenti-shRhoAscr) was generated using the same vector and the following oligo DNA pair: 5′-CGCGTGGCAAATCTTCTAGTCTATTTCAAGAGAATAGACTAGAAGATTTGCCTTTTTTA-3′ and 5′-CGCGTAAAAAAGGCAAATCTTCTAGTCTATTCTCTTGAAATAGACTAGAAGATTTGCCA-3′.…”

Section: Methodsmentioning

confidence: 99%

Activating transcription factor 4 (ATF4) modulates post-synaptic development and dendritic spine morphology

Liu

Pasini

Shelanski

et al. 2014

Front. Cell. Neurosci.

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The ubiquitously expressed activating transcription factor 4 (ATF4) has been variably reported to either promote or inhibit neuronal plasticity and memory. However, the potential cellular bases for these and other actions of ATF4 in brain are not well-defined. In this report, we focus on ATF4's role in post-synaptic synapse development and dendritic spine morphology. shRNA-mediated silencing of ATF4 significantly reduces the densities of PSD-95 and GluR1 puncta (presumed markers of excitatory synapses) in long-term cultures of cortical and hippocampal neurons. ATF4 knockdown also decreases the density of mushroom spines and increases formation of abnormally-long dendritic filopodia in such cultures. In vivo knockdown of ATF4 in adult mouse hippocampal neurons also reduces mushroom spine density. In contrast, ATF4 over-expression does not affect the densities of PSD-95 puncta or mushrooom spines. Regulation of synaptic puncta and spine densities by ATF4 requires its transcriptional activity and is mediated at least in part by indirectly controlling the stability and expression of the total and active forms of the actin regulatory protein Cdc42. In support of such a mechanism, ATF4 silencing decreases the half-life of Cdc42 in cultured cortical neurons from 31.5 to 18.5 h while knockdown of Cdc42, like ATF4 knockdown, reduces the densities of mushroom spines and PSD-95 puncta. Thus, ATF4 appears to participate in neuronal development and plasticity by regulating the post-synaptic development of synapses and dendritic mushroom spines via a mechanism that includes regulation of Cdc42 levels.

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Delayed Intrathecal Delivery of RhoA siRNA to the Contused Spinal Cord Inhibits Allodynia, Preserves White Matter, and Increases Serotonergic Fiber Growth

Cited by 31 publications

References 54 publications

Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model

Activating transcription factor 4 (ATF4) modulates post-synaptic development and dendritic spine morphology

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