Long-Term Gene-Silencing Effects of siRNA Introduced by Single-Cell Electroporation into Postmitotic CNS Neurons

Tanaka, Masahiko; Asaoka, Minami; Yanagawa, Yuchio; Hirashima, Naohide

doi:10.1007/s11064-011-0474-6

Cited by 10 publications

(4 citation statements)

References 19 publications

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“…The prosurvival effect of DLK inhibition persisted for at least 3 wk. We found that Dlk mRNA levels stayed low throughout this period, which is consistent with reports that siRNA knockdown in postmitotic neurons can be long-lived (28,29).…”

Section: Dlk Down-regulation Promotes Long-term Survival and Function Ofsupporting

confidence: 92%

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Welsbie

Yang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

237

307

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Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.G laucoma is the leading cause of irreversible blindness worldwide (1). It is a neurodegenerative disease in which vision loss is caused by the axonal injury and death of retinal ganglion cells (RGCs) (2), the projection neurons that process and transmit vision from the retina to the brain. Current therapies (i.e., surgery, laser, and eye drops) all act by lowering intraocular pressure (IOP). However, pressure reduction can be difficult to achieve, and even with significant pressure lowering, RGC loss can continue. Efforts have therefore been made to develop neuroprotective agents that would complement IOP-lowering therapies by directly inhibiting the RGC cell death process (3, 4). However, no neuroprotective agent has yet been approved for clinical use.Protein kinases provide attractive targets for the development of neuroprotective agents. A number of kinases, including cyclindependent kinases, death-associated protein kinases, JNK1-3, MAPKs, and glycogen synthase kinase-3β, are involved in neuronal cell death (5-12). An additional attraction is that protein kinases are readily druggable. The pharmacology and medicinal chemistry of kinase inhibitors are well-developed, with kinases now being the most important class of drug targets after G protein-coupled receptors (13). Although the primary clinical use of kinase inhibitors continues to be as antineoplastic agents, increasing attention is being paid to their use in other areas (14,15).To identify, in a comprehensive and unbiased manner, kinases that could serve as targets for neuroprotective glaucoma therapy, we screened the entire mouse kinome for kinases whose inhibition promotes RGC survival. For this screen, we develope...

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Section: Dlk Down-regulation Promotes Long-term Survival and Function Ofsupporting

confidence: 92%

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Welsbie

Yang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

237

307

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“…The method of single‐cell electroporation of siRNA was largely described previously (Tanaka et al, , ; Miyata et al, ; Tanaka, ). Micropipettes were pulled on a micropipette puller (PB‐7; Narishige, Tokyo, Japan) using 1.2 mm o.d.…”

Section: Methodsmentioning

confidence: 99%

“…It enables us to transfer siRNAs and inhibit the expression of target genes in specific neurons in an environment composed of different types of cell (Boudes et al, ; Tanaka et al, ; Miyata et al, ; Tanaka, ). Because the knockdown effects by this method continue for at least 14 days after electroporation in postmitotic neurons (Tanaka et al, ), it can be applied to investigations of the molecular mechanisms of dendrite formation occurring over a period of several days or weeks.…”

Section: Introductionmentioning

confidence: 99%

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

Ohashi

Sakata

Naito

et al. 2013

Developmental Neurobiology

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Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

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“…The temporal alterations in the GFP fluorescence (in the same electroporated cells) were observed for 4–14 days via repeated imaging ( Figure 15 ). Furthermore, they checked the dependency of concentration for specific gene silencing and the non-specific off-target effects of siRNA inserted through this method, showing that the effects were present at least up to 14 days, yet differed between neuronal cell types [ 122 , 168 ].…”

Section: Single-neuron Transfection Methodsmentioning

confidence: 99%

A Single-Neuron: Current Trends and Future Prospects

Gupta

Balasubramaniam

Chang

et al. 2020

Cells

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The brain is an intricate network with complex organizational principles facilitating a concerted communication between single-neurons, distinct neuron populations, and remote brain areas. The communication, technically referred to as connectivity, between single-neurons, is the center of many investigations aimed at elucidating pathophysiology, anatomical differences, and structural and functional features. In comparison with bulk analysis, single-neuron analysis can provide precise information about neurons or even sub-neuron level electrophysiology, anatomical differences, pathophysiology, structural and functional features, in addition to their communications with other neurons, and can promote essential information to understand the brain and its activity. This review highlights various single-neuron models and their behaviors, followed by different analysis methods. Again, to elucidate cellular dynamics in terms of electrophysiology at the single-neuron level, we emphasize in detail the role of single-neuron mapping and electrophysiological recording. We also elaborate on the recent development of single-neuron isolation, manipulation, and therapeutic progress using advanced micro/nanofluidic devices, as well as microinjection, electroporation, microelectrode array, optical transfection, optogenetic techniques. Further, the development in the field of artificial intelligence in relation to single-neurons is highlighted. The review concludes with between limitations and future prospects of single-neuron analyses.

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Long-Term Gene-Silencing Effects of siRNA Introduced by Single-Cell Electroporation into Postmitotic CNS Neurons

Cited by 10 publications

References 19 publications

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

A Single-Neuron: Current Trends and Future Prospects

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