Action of triton X-100 on ultrastructure and membrane-bound enzymes of isolated nerve endings from rat brain

Fiszer, Sara; Robertis, E. De

doi:10.1016/0006-8993(67)90217-x

Cited by 95 publications

(15 citation statements)

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“…We utilized a biochemical approach that isolates neuronal subcellular fractions using filtration and detergent solubility to examine K v 1.1 and K v 1.2 expression in a PSD-enriched fraction. It is well established that resident postsynaptic density (PSD) proteins are not soluble in the detergent Triton X-100 (Fiszer and Robertis, 1967). Thus, we reasoned that we could determine K v 1.1 and 1.2 by biochemically isolating the lysate (total), Triton X-soluble (dendrites and axons), and Triton X-insoluble (PSD) as outlined in Figure 2 of Niere et al (2016), (Fiszer and Robertis, 1967; Cohen et al, 1977; Rao and Steward, 1991; Villasana et al, 2006).…”

Section: Mtorc1-mediated Positive Feedback Regulation Of Local Dendrmentioning

confidence: 99%

“…It is well established that resident postsynaptic density (PSD) proteins are not soluble in the detergent Triton X-100 (Fiszer and Robertis, 1967). Thus, we reasoned that we could determine K v 1.1 and 1.2 by biochemically isolating the lysate (total), Triton X-soluble (dendrites and axons), and Triton X-insoluble (PSD) as outlined in Figure 2 of Niere et al (2016), (Fiszer and Robertis, 1967; Cohen et al, 1977; Rao and Steward, 1991; Villasana et al, 2006). Summarily, we first assessed the purity of fractionated samples by Western blotting for well-characterized resident proteins: postsynaptic density protein of 95 kD (PSD95, PSD marker) and synapsin 1 (presynaptic marker).…”

Section: Mtorc1-mediated Positive Feedback Regulation Of Local Dendrmentioning

confidence: 99%

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mTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways

Niere

Raab‐Graham

2017

Front. Cell. Neurosci.

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The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) serves as a regulator of mRNA translation. Recent studies suggest that mTORC1 may also serve as a local, voltage sensor in the postsynaptic region of neurons. Considering biochemical, bioinformatics and imaging data, we hypothesize that the activity state of mTORC1 dynamically regulates local membrane potential by promoting and repressing protein synthesis of select mRNAs. Our hypothesis suggests that mTORC1 uses positive and negative feedback pathways, in a branch-specific manner, to maintain neuronal excitability within an optimal range. In some dendritic branches, mTORC1 activity oscillates between the “On” and “Off” states. We define this as negative feedback. In contrast, positive feedback is defined as the pathway that leads to a prolonged depolarized or hyperpolarized resting membrane potential, whereby mTORC1 activity is constitutively on or off, respectively. We propose that inactivation of mTORC1 increases the expression of voltage-gated potassium alpha (Kv1.1 and 1.2) and beta (Kvβ2) subunits, ensuring that the membrane resets to its resting membrane potential after experiencing increased synaptic activity. In turn, reduced mTORC1 activity increases the protein expression of syntaxin-1A and promotes the surface expression of the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA)-type subunit 1 (GluN1) that facilitates increased calcium entry to turn mTORC1 back on. Under conditions such as learning and memory, mTORC1 activity is required to be high for longer periods of time. Thus, the arm of the pathway that promotes syntaxin-1A and Kv1 protein synthesis will be repressed. Moreover, dendritic branches that have low mTORC1 activity with increased Kv expression would balance dendrites with constitutively high mTORC1 activity, allowing for the neuron to maintain its overall activity level within an ideal operating range. Finally, such a model suggests that recruitment of more positive feedback dendritic branches within a neuron is likely to lead to neurodegenerative disorders.

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Section: Mtorc1-mediated Positive Feedback Regulation Of Local Dendrmentioning

confidence: 99%

Section: Mtorc1-mediated Positive Feedback Regulation Of Local Dendrmentioning

confidence: 99%

mTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways

Niere

Raab‐Graham

2017

Front. Cell. Neurosci.

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“…Through a series of filtration steps and detergent solubilization, we isolated fractions of synaptoneurosomes that were soluble (supernatant, S, containing neuronal processes void of the PSD) and insoluble (pellet, P, PSD) in the detergent, triton X-100 (TX-100) ( Fig. 2A, illustration of biochemical isolation) (70,71). We used this modified approach for enriching PSD to allow us to identify promising proteins that directly interact with resident proteins of PSD (see Experimental Procedures).…”

Section: Psd Proteome Is Labile During Brief Rapamycin Exposure and Cmentioning

confidence: 99%

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Niere

Namjoshi²,

Song

et al. 2016

Molecular & Cellular Proteomics

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Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) 1 is a serine/threonine protein kinase that is highly Author contributions: FN and KRG designed research. FN and SN conducted experiments and analyzed data. SN performed bioinformatics analyses. ES and YM conducted mass spectrometry analyses. GS facilitated mass spectrometry experiments and provided technical advice. GAD and BVZ provided the virus and performed stereotaxic injections. FN, SN, and KRG wrote the manuscript. 1 The abbreviations used are: mTORC1, mechanistic/mammalian target of rapamycin complex 1; AD, Alzheimer's disease; AHA, azidohomoalanine; APP, amyloid precursor protein; ASD, autism spectrum disorder; BONCAT, bioorthogonal noncanonical amino acid

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“…The molecular characterization of the synaptic complex is dependent on the availability of procedures for the preparation of subcellular fractions enriched in one or more synaptic structures. Following the observation of Fiszer and deRobertis (1967) that extraction of synaptic membranes with Triton X-100 leaves an insoluble residue that is enriched in structures derived from the synaptic junctional complex, several laboratories reported methods for the isolation of fractions containing purified synaptic junctions (Cotman and Taylor, 1972;Davis and Bloom, 1973;Therien and Mushynski, 1976) and postsynaptic densities (PSDs) (Cotman et al, 1974;Cohen et al, 1977;Matus and Taff-Jones, 1978). Biochemical analysis of these fractions has provided a considerable amount of information relating J .…”

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confidence: 99%

Biochemical and Morphological Comparison of Postsynaptic Densities Prepared from Rat, Hamster, and Monkey Brains by Phase Partitioning

Gurd

Gordon‐Weeks

Evans

1982

Journal of Neurochemistry

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A new procedure for the preparation of postsynaptic densities (PSDs) is described. A synaptic membrane fraction was homogenized in an aqueous two-phase polymer system containing Poly(ethylene glycol) 6000 (5% wt/wt) and Dextran T500 (6% wt/wt) containing 1% 1-o-n-octyl-beta-D-glucoside. Following a brief centrifugation to separate the phases, highly purified PSDs banded at the interface of the two phases. Using this procedure PSDs have been isolated from rat and hamster cerebral cortex and from the frontal cortex, cerebral cortex, hippocampus, and pooled caudate/putamen regions of Macaca mulatta Rhesus monkeys. The isolated PSDs appeared as curved bars when sectioned or as discs when viewed en face in the electron microscope. The hamster PSDs were associated with large numbers of small rod-like structures 4.5 nm thick and 28 nm long. Similar structures were present, although in fewer numbers, in the rat and monkey preparations. Polyacrylamide gel electrophoresis showed that the PSDs contained a complex population of proteins with major components having molecular weights of 180,000, 130,000, 110,000, 94,000, 65,000, 60,000, and 51,000. Reaction of polyacrylamide gels with 125I-concanavalin A (Con A) identified two major (apparent Mr 180,000 and 130,000) and three minor (apparent Mr 230,000, 145,000, and 110,000) Con A-binding glycoproteins in the PSD fractions. Although some quantitative variation between species and brain regions was apparent, the overall protein and glycoprotein composition was similar for all fractions studied.

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Action of triton X-100 on ultrastructure and membrane-bound enzymes of isolated nerve endings from rat brain

Cited by 95 publications

References 20 publications

mTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways

mTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Biochemical and Morphological Comparison of Postsynaptic Densities Prepared from Rat, Hamster, and Monkey Brains by Phase Partitioning

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