High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

Jiang, Min; Deng, Lin; Chen, G

doi:10.1038/sj.gt.3302305

Cited by 64 publications

(49 citation statements)

References 23 publications

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“…We used lentiviral plasmids containing shPrP and empty vector constructs as described 9 . Lentiviruses were generated using DNA/Ca 21 phosphate transfection of HEK293 cells 33 . Additional stocks of virus were generated by GenTarget and titre determined using fluorescence-activated cell sorting (BD FACS Calibur).…”

Section: Methodsmentioning

confidence: 99%

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

Moreno

Radford

Peretti

et al. 2012

Nature

562

617

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The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the a-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2a-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases 1-4 , but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2a-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2a-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2a-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2a-P dephosphorylation 5 , increased eIF2a-P levels, exacerbating neurotoxicity and significantly reducing survival in priondiseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.Neurodegenerative diseases pose an ever-increasing challenge for society and health care systems worldwide, but their molecular pathogenesis is still largely unknown and no curative treatments exist. Alzheimer's (AD), Parkinson's (PD) and prion diseases are separate clinical and pathological conditions, but it is likely they share common mechanisms leading to neuronal death. Mice with prion disease show misfolded prion protein (PrP) accumulation and develop extensive neurodegeneration (with profound neurological deficits), in contrast to mouse models of AD or PD, in which neuronal loss is rare. Uniquely therefore, prion-infected mice allow access to mechanisms linking protein misfolding with neuronal death. Prion replication involves the conversion of cellular PrP, PrP C , to its misfolded, aggregating conformer, PrP Sc , a process leading ultimately to neurodegeneration 6 . We have previously shown rescue of neuronal loss and reversal of early cognitive and morphological changes in prion-infected mice by depleting PrP in neurons, preventing prion replication and ab...

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Section: Methodsmentioning

confidence: 99%

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

Moreno

Radford

Peretti

et al. 2012

Nature

562

617

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“…Neuronal Transfection-Hippocampal cultures were transfected using the calcium phosphate precipitate technique as previously described (18). Briefly, DNA/Ca 2ϩ -phosphate precipitate was prepared by diluting (per coverslip) 2 l of 1 mg/ml plasmid DNA in 20.5 l of filter-sterilized TE (10 mM Tris-Cl, 1 mM EDTA, pH 7.3) followed by addition of 2.5 l of filtersterilized HEPES-buffered CaCl 2 (2.5 M CaCl 2 , 10 mM HEPES, pH 7.2).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Dysfunction and Dendritic Beading during Neuronal Toxicity

Greenwood

Mizielinska

Frenguelli

et al. 2007

Journal of Biological Chemistry

106

102

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Mitochondrial dysfunction (depolarization and structural collapse), cytosolic ATP depletion, and neuritic beading are early hallmarks of neuronal toxicity induced in a variety of pathological conditions. We show that, following global exposure to glutamate, mitochondrial changes are spatially and temporally coincident with dendritic bead formation. During oxygen-glucose deprivation, mitochondrial depolarization precedes mitochondrial collapse, which in turn is followed by dendritic beading. These events travel as a wave of activity from distal dendrites toward the neuronal cell body. Despite the spatiotemporal relationship between dysfunctional mitochondria and dendritic beads, mitochondrial depolarization and cytoplasmic ATP depletion do not trigger these events. However, mitochondrial dysfunction increases neuronal vulnerability to these morphological changes during normal physiological activity. Our findings support a mechanism whereby, during glutamate excitotoxicity, Ca 2؉ influx leads to mitochondrial depolarization, whereas Na ؉ influx leads to an unsustainable increase in ATP demand (Na ؉ ,K ؉ -ATPase activity). This leads to a drop in ATP levels, an accumulation of intracellular Na ؉ ions, and the subsequent influx of water, leading to microtubule depolymerization, mitochondrial collapse, and dendritic beading. Following the removal of a glutamate challenge, dendritic recovery is dependent upon the integrity of the mitochondrial membrane potential, but not on a resumption of ATP synthesis or Na ؉ ,K ؉ -ATPase activity. Thus, dendritic recovery is not a passive reversal of the events that induce dendritic beading. These findings suggest that the degree of calcium influx and mitochondrial depolarization inflicted by a neurotoxic challenge, determines the ability of the neuron to recover its normal morphology.Mitochondrial dysfunction, dendritic spine remodeling, and the formation of neuritic beads are early hallmarks of neuronal toxicity induced in a variety of pathological conditions (1-6). In particular, in Alzheimer disease, the existence of extracellular amyloid plaques and intracellular neurofibrillary tangles have been associated with neuritic beading, spine loss, neurite breakage, and shaft atrophy (7-10).An understanding of the importance of mitochondrial dysfunction in neurological diseases continues to grow. Mitochondria are highly dynamic organelles with their trafficking and elongated morphology being regulated by neuronal (11, 12) and mitochondrial (13) activity. It appears that mitochondrial transport, morphology, and function are affected by physiological processes such as synaptic transmission and pathological processes such as excitotoxicity.Despite the fact that mitochondrial dysfunction and dendritic beading are both initiated by glutamate excitotoxicity, these events are believed to be independent due to their distinct requirements for Ca 2ϩ (1-4, 6, 14) versus Na ϩ (2, 3) influx. Moreover, dendritic beading occurs much more rapidly than Ca 2ϩ -dependent excitotoxic cell death (2, 3). ...

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“…PC12 cells were cultured in RPMI 1640 medium (Invitrogen) containing 2 mM L-glutamine and penicillin and streptomycin. Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol or calcium phosphate method (Clontech) as previously described (29). Neurons were transfected at least 3 days before fixation and immunostaining.…”

Section: Methodsmentioning

confidence: 99%

Involvement of Myosin Vb in Glutamate Receptor Trafficking

Lisé

Wong

Trinh

et al. 2006

Journal of Biological Chemistry

118

107

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Myosin V motors mediate cargo transport; however, the identity of neuronal molecules transported by these proteins remains unknown. Here we show that myosin Vb is expressed in several neuronal populations and associates with the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor subunit GluR1. In developing hippocampal neurons, expression of the tail domain of myosin Vb, but not myosin Va, enhanced GluR1 accumulation in the soma and reduced its surface expression. These changes were accompanied by reduced GluR1 clustering and diminished frequency of excitatory but not inhibitory synaptic currents. Similar effects were observed upon expression of full-length myosin Vb lacking a C-terminal region required for binding to the small GTPase Rab11. In contrast, mutant myosin Vb did not change the localization of several other neurotransmitter receptors, including the glutamate receptor subunit NR1. These results reveal a novel mechanism for the transport of a specific glutamate receptor subunit in neurons mediated by a member of the myosin V family.Proper sorting and transport of excitatory neurotransmitter receptors and associated proteins is essential for neuronal activity and plasticity. Recent studies have identified several proteins that regulate clustering of neurotransmitter receptors at the synapse (1). However, it remains unknown what proteins mediate sorting and delivery of receptors from the soma to postsynaptic sites. Molecular motors that regulate cargo trafficking on both actin filaments and microtubules have been implicated in initial transport and delivery to specific subcellular sites (2, 3). In particular, class V of unconventional myosins is actin-based motors thought to regulate trafficking of organelles and associated proteins in neuronal cells (2, 4).Three known members of the myosin V family have been detected in brain extracts. The most studied member, myosin Va, is widely expressed in the brain (5). Dilute mice, which possess mutation in the myosin Va gene, suffer from impaired melanosome transport and severe seizures and die within 2-3 weeks after birth (6). These observations suggest that alteration in the transport of important yet unknown cargos contributed to the observed defects in neuronal function. In neurons, myosin Va is enriched at the postsynaptic density (PSD) 4 of excitatory synapses (7) and associates with the scaffolding guanylate kinase domain-associated protein through interaction with the dynein light chain (8). The association of guanylate kinase domain-associated protein with the postsynaptic density protein-95 (PSD-95), a protein involved in glutamate receptor clustering, may functionally couple these proteins to myosin Va (1, 9).Myosin Vb and myosin Vc are two additional members of the myosin V family that are also expressed in the brain; however, their exact localization in neurons remains unclear (10, 11). All of these motors share ϳ42% identity and contain a conserved N-terminal motor domain followed by a coiled-coil region and a globular C-termi...

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High Ca2+-phosphate transfection efficiency enables single neuron gene analysis

Cited by 64 publications

References 23 publications

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

Sustained translational repression by eIF2α-P mediates prion neurodegeneration

Mitochondrial Dysfunction and Dendritic Beading during Neuronal Toxicity

Involvement of Myosin Vb in Glutamate Receptor Trafficking

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