Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons

Gascón, Sergio; Paez-Gomez, Juan A; Dı́az-Guerra, Margarita; Scheiffele, Peter; Scholl, Francisco G.

doi:10.1016/j.jneumeth.2007.09.023

Cited by 75 publications

(84 citation statements)

References 31 publications

(39 reference statements)

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“…Acute brain slices, dissociated neuronal cultures, lentiviruses, and immunoelectron microscopy were performed as described previously (8,11,24,25). Detailed materials and methods are described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…We then assessed rescue of this phenotype by introducing either NL1 or chimeric NLs into the hippocampus of NL1 KO animals by stereotaxic injection of recombinant, dual-promoter lentiviruses (Fig. S4A) (24). Reintroduction of WT NL1 and NL1-2 but not NL2-1 restored recovery of NMDAR EPSCs that represents synaptic incorporation of NMDARs (Fig.…”

Section: Synaptic Incorporation and Retention Of Nmdars Controlled Bymentioning

confidence: 99%

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Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling

Budreck

Kwon

Jung

et al. 2012

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

Self Cite

162

161

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Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cisinteractions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties.synapse | neurotranmitter receptor | neurexin N MDA receptors (NMDARs) are key regulators of the development of neural circuits and synaptic plasticity (1, 2). In humans, perturbation of NMDAR function results in psychotic conditions, and genetic animal models with altered NMDAR activity exhibit phenotypes related to cognitive disorders such as schizophrenia and autism (3-5). Activity-dependent recruitment of NMDARs to synapses controls certain forms of synaptic plasticity (6, 7). However, the molecular mechanisms underlying the recruitment and physical tethering of NMDAR complexes at synapses are incompletely understood.Neuroligin-1 (NL1), one of four postsynaptic neuroligin adhesion molecules (NL1, 2, 3, 4), contributes to NMDAR regulation (8, 9). In cultured neurons, overexpression of NL1 promotes clustering of synaptic NMDARs (8), and NL1 KO mice show decreases in NMDAR-dependent excitatory postsynaptic currents (NMDAR EPSCs) (9-11). A major question in understanding neuroligin function is how specific isoforms couple to specific neurotransmitter receptors (12, 13). NMDARs were recovered in coimmunoprecipitations with NL proteins, indicating a potential complex formation, although in those experiments, no NL isoform-specificity was apparent (14). One candidate link between NLs and glutamate receptors is through postsynaptic scaffolding molecules such as postsynaptic density 95 (PSD95) (15, 16). However, all NL isoforms contain PSD95 binding sites, and NMDARs and PSD95 were recruited to NL1 with different time courses (14).Our results demonstrate that NL1 controls synaptic abundance of NMDAR via NL1-specific extracellular determinants. Loss of these interactions results in impairment of NMDAR-mediated transmission and synaptic plasticity. Our findings uncover an unexpected mode of NL1-NMDAR coupling and demonstrate a key role for the NL1 adhesion protein in the physical incorporation and retention of NMDAR at glutamatergic synapses.Results NL1-Specific Recruitment of NMDARs Does Not Require PSD95. We examined the specificity of molecular coupling of NL isoforms (NL1, 2, 3) to NMDARs by NL overexpression in cultured hippocampal neurons. NL1 increased the density of clusters of the NMDAR subunits GluN1, GluN2A, and GluN...

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Section: Methodsmentioning

confidence: 99%

Section: Synaptic Incorporation and Retention Of Nmdars Controlled Bymentioning

confidence: 99%

Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling

Budreck

Kwon

Jung

et al. 2012

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

Self Cite

162

161

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“…The DNA binding domain (DBD) of TFs CREB, SRF, EGR1, and FOS was fused with the acidic transactivation domain of viral protein 16 (VP16) of herpes simplex virus (Flint and Shenk, 1997). These chimeric constructs, as well as the coding sequence of A-CREB (Ahn et al, 1998), were cloned into the Synapsin promoter-bearing lentiviral vector LenLox 3.7 (Gascó n et al, 2008). Production of lentiviral pseudovirions was performed as described by Gascó n et al (2008) with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

cAMP Response Element-Binding Protein Is a Primary Hub of Activity-Driven Neuronal Gene Expression

Benito¹,

Valor²,

Jiménez-Minchan³

et al. 2011

J. Neurosci.

104

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Long-lasting forms of neuronal plasticity require de novo gene expression, but relatively little is known about the events that occur genome-wide in response to activity in a neuronal network. Here, we unveil the gene expression programs initiated in mouse hippocampal neurons in response to different stimuli and explore the contribution of four prominent plasticity-related transcription factors (CREB, SRF, EGR1, and FOS) to these programs. Our study provides a comprehensive view of the intricate genetic networks and interactions elicited by neuronal stimulation identifying hundreds of novel downstream targets, including novel stimulus-associated miRNAs and candidate genes that may be differentially regulated at the exon/promoter level. Our analyses indicate that these four transcription factors impinge on similar biological processes through primarily non-overlapping gene-expression programs. Meta-analysis of the datasets generated in our study and comparison with publicly available transcriptomics data revealed the individual and collective contribution of these transcription factors to different activity-driven genetic programs. In addition, both gain-and loss-of-function experiments support a pivotal role for CREB in membrane-to-nucleus signal transduction in neurons. Our data provide a novel resource for researchers wanting to explore the genetic pathways associated with activity-regulated neuronal functions.

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“…In the absence of Tet or its derivatives such as doxycycline (Dox), the tTA binds the Tet-response element (TRE) that is composed of seven tandem copies of Tet operator (tetO) sequences and placed upstream of the CMV minimal promoter (Baron & Bujard, 2000). This type of original Tet-regulated system, called Tet-off system, has been incorporated into HIV-1-derived lentiviral vectors (Gascon et al, 2008;Haack et al, 2004;Kafri et al, 2000;Vigna et al, 2002) (Fig. 4A).…”

Section: Tetracycline-regulated Systemmentioning

confidence: 99%

“…This can be a bottleneck, particularly in relevant applications of the Tet-regulated lentiviral vector systems in clinical use. Single vector systems that contain the entire regulatable component in a unique vector is one of the solutions to guarantee simultaneous expression of the two expression units in the target gene, and these have indeed been established (Gascon et al, 2008;Kafri et al, 2000;Szulc et al, 2006;Vogel et al, 2004).…”

Section: Tetracycline-regulated Systemmentioning

confidence: 99%

Gene Regulatable Lentiviral Vector System

Suzuki¹,

Suzuki²

2011

Viral Gene Therapy

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Dual-promoter lentiviral vectors for constitutive and regulated gene expression in neurons

Cited by 75 publications

References 31 publications

Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling

Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling

cAMP Response Element-Binding Protein Is a Primary Hub of Activity-Driven Neuronal Gene Expression

Gene Regulatable Lentiviral Vector System

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