In vivo Inhibition of Hippocampal Ca2+/Calmodulin-Dependent Protein Kinase II by RNA Interference

Babcock, Alex M.; Standing, D.; Bullshields, K.; Schwartz, Elizabeth Ann.; Paden, Charles M.; Poulsen, David J.

doi:10.1016/j.ymthe.2005.02.016

Cited by 26 publications

(28 citation statements)

References 30 publications

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“…In fact, several recent reports have demonstrated the feasibility of such manipulations in the brain (20)(21)(22)(23). Viral vector-mediated RNAi technology provides several advantages over existing transgenic techniques.…”

Section: Discussionmentioning

confidence: 99%

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

Musatov

Chen

Pfaff

et al. 2006

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

200

154

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Estrogen receptor ␣ (ER␣) plays a major role in the regulation of neuroendocrine functions and behaviors by estrogens. Although the generation of ER␣ knockout mice advanced our knowledge of ER␣ functions, gene deletion using this method is global and potentially confounded by developmental consequences. To achieve a site-specific knockdown of ER␣ in the normally developed adult brain, we have generated an adeno-associated virus vector expressing a small hairpin RNA targeting ER␣. After bilateral injection of this vector into the hypothalamic ventromedial nucleus in ovariectomized female mice, expression levels of ER␣ as well as the estrogen-inducible progesterone receptor were profoundly reduced despite the continued presence of this receptor elsewhere in the brain. Functionally, silencing of ER␣ in the ventromedial nucleus abolished female proceptive and receptive sexual behaviors while enhancing rejection behavior. These results provide evidence that adeno-associated virus-mediated long-term knockdown of genes can be used to delineate their effects on complex behaviors in discrete brain regions.adeno-associated virus ͉ lordosis ͉ RNA interference ͉ viral vector E strogen elicits many important biochemical responses in the brain, yet the respective actions of the two estrogen receptors, ER␣ and ER␤, in mediating these effects are not completely understood (1-3). Generation of ER␣ knockout (ERKO) mice has shed light on some of the functions of ER␣ signaling. Of particular interest is estrogen action in the ventromedial nucleus of hypothalamus (VMN), which appears to be critical for several behaviors, including female reproductive behavior. VMN is a key player in a neural circuit controlling the execution of lordosis, a hormone-dependent reflexive posture exhibited by sexually receptive female rodents in response to male mounting (4). Analysis of homozygous ERKO females revealed that they completely lack lordosis behavior. Furthermore, these mice are also deficient in sexual behavioral interactions that precede the lordosis response. They are extremely aggressive and rejective toward attempted mounts by male mice, thus preventing copulation (5-7). In dramatic contrast, reproductive behaviors of ER␤ knockout females are undistinguishable from those of wild-type mice (3).Although these studies identified ER␣ as a gene essential for several mammalian behaviors, it remained unclear as to which of these effects are due to a mere absence of this receptor in the mature neurons and which deficits result from the lack of ER␣ expression during development or as a consequence of genetic compensation. Moreover, recent findings indicate a potential confound in the design of ERKO mice, because these animals express an abnormal splicing variant of ER␣ (8). This truncated form of the receptor retains both the DNA-and ligand-binding domains and is able to mediate, albeit far less efficiently, at least some estrogen effects. Therefore, somatic gene knockdown in individual nuclei of normally developed brain through RNA interference...

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Section: Discussionmentioning

confidence: 99%

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

Musatov

Chen

Pfaff

et al. 2006

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

200

154

View full text Add to dashboard Cite

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“…In addition, AAV5 was efficiently transduced into T cells and macrophages ( Fig. 3 and 4), which may be useful for gene therapy of diseases involving hematopoietic cells (4,26,43,57,59,69).…”

Section: Discussionmentioning

confidence: 99%

Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 forDendritic Cells

Xin

Mizukami

Urabe

et al. 2006

J Virol

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The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cellmediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.

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“…Several recent reports have demonstrated the utility of rAAV vectors for gene silencing in the CNS using RNAi (Hommel et al, 2003;Babcock et al, 2005;Machida et al, 2006;Musatov et al, 2006). rAAV vectors can be produced in high titers and can transduce postmitotic neurons in the brain and SCDH in a highly spatially localized manner (Kaplitt et al, 1994;Kaspar et al, 2002;South et al, 2003).…”

Section: Figmentioning

confidence: 99%

Design and Evaluation of Small Interfering RNAs That Target Expression of the N-Methyl-d-aspartate Receptor NR1 Subunit Gene in the Spinal Cord Dorsal Horn

Garraway

Xu²,

Inturrisi³

2007

J Pharmacol Exp Ther

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NR1 is an essential subunit of the N-methyl-D-aspartate (NMDA) receptor, which at the spinal level is involved in injuryinduced pain hypersensitivity and morphine tolerance. An in vitro luciferase assay was used to identify candidate and control (inactive) short interfering RNA (siRNA) sequences that are expressed by a recombinant adeno-associated virus (rAAV) plasmid. rAAV vectors targeting the NR1 subunit were prepared that express active or control (mismatch) siRNA sequences and injected into the mouse spinal cord dorsal horn (SCDH). Three weeks after vector administration, green fluorescent protein labeling of the ipsilateral SCDH confirmed the spatial localization of the viral transduction. Active siRNAs resulted in a 60 to 75% knockdown of NR1 mRNA and protein in the area of the virus injection. The spatial knockdown persisted for at least 6 months after a single administration of the vector. Neither the active nor the mismatch siRNAs resulted in cellular toxicity as measured by nuclear staining and cell integrity. The vectorderived knockdown of NR1 expression in SCDH did not alter acute thermal or mechanical stimulus paw-withdrawal thresholds. However, the vector-derived siRNA prevented the mechanical allodynia measured at 24 and 48 h after injection into the paw of the inflammatory agent, Complete Freund's adjuvant. These results demonstrate that vector-derived siRNAs can be used to produce an in vivo spatial knockdown of the expression and function of the NMDA receptor that is confined to the ipsilateral SCDH. Vector-derived siRNAs may have therapeutic potential for the management of injury-induced pain resulting from the activation of NMDA receptors in the SCDH.RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Because it allows a sequence-specific inhibition of gene expression, it is being widely applied in biology, including in vivo applications (Fire et al., 1998;Dorsett and Tuschl, 2004;Dykxhoorn et al., 2006).RNAi is often more potent and longer lasting than other nucleic acid-based approaches and, compared to gene targeting by homologous recombination, is less expensive and timeconsuming and not limited to a particular mammalian species (i.e., the mouse) (Dorsett and Tuschl, 2004;Dykxhoorn et al., 2006). A key feature of this approach is that small doublestranded RNAs (small interfering RNAs, siRNAs) are generated by the action of an RNase-III-like enzyme called Dicer from a larger double-stranded RNA precursor or from short hairpin (sh) RNAs, which can be expressed from plasmids or viral vectors (Lieberman et al., 2003;Dorsett and Tuschl, 2004). mRNA degradation occurs when the antisense strand of the siRNA directs the RNA-induced silencing complex that contains the RNA endonuclease Ago2 to cleave the complementary sequence of the target mRNA (Dykxhoorn et al., 2006). Silencing by RNAi is typically incomplete-a knockdown rather than a knockout. Similar to other nucleic acidbased methods, any gene is a potential ...

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In vivo Inhibition of Hippocampal Ca2+/Calmodulin-Dependent Protein Kinase II by RNA Interference

Cited by 26 publications

References 30 publications

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

RNAi-mediated silencing of estrogen receptor α in the ventromedial nucleus of hypothalamus abolishes female sexual behaviors

Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 forDendritic Cells

Design and Evaluation of Small Interfering RNAs That Target Expression of the N-Methyl-d-aspartate Receptor NR1 Subunit Gene in the Spinal Cord Dorsal Horn

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