Protein Synthesis in a Synaptosomal Fraction from Squid Brain

Crispino, Marianna; Castigli, Emilia; Perrone-Capano, Carla; Martin, Rainer de; Menichini, E.; Kaplan, Barry B.; Giuditta, Antonio

doi:10.1006/mcne.1993.1046

Cited by 48 publications

(53 citation statements)

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“…To ensure that the amplicons generated by qRT-PCR were indeed derived from polysomal RNA, we purified polysomes with an isolation buffer containing EDTA, a Mg 2+ chelating agent known to dissociate polysomes. Under these isolation conditions, the dissociated polysomes are unable to sediment through heavy sucrose (Giuditta et al 1991;Crispino et al 1993). The qRT-PCR tdTomato mRNA levels in the cell bodies of SCG neurons determined by qRT-PCR 72 h after DNA transfection.…”

Section: Discussionmentioning

confidence: 99%

“…Addition of ethylenediaminetetraacetic acid (EDTA), a chelating agent known to dissociate polysomes, markedly diminished the PCR-generated signals. Under these experimental conditions, the dissociated polysomes are incapable of sedimenting through 2.0 M sucrose and hence were eliminated from the polysome fraction (Giuditta et al 1991;Crispino et al 1993Crispino et al , 1997. Less than 2% of total TH RNA co-sedimented with the polysome fraction in the presence of EDTA.…”

Section: Th Is Synthesized In Distal Axons Of Primary Noradrenergic Smentioning

confidence: 99%

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The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

Gervasi

Scott

Aschrafi

et al. 2016

RNA

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Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3 ′ UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.

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Section: Discussionmentioning

confidence: 99%

Section: Th Is Synthesized In Distal Axons Of Primary Noradrenergic Smentioning

confidence: 99%

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

Gervasi

Scott

Aschrafi

et al. 2016

RNA

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“…T wo 4 ml aliquots of the synaptosomal fraction were incubated at 18 -20°C for 1 hr in a medium containing 0.84 M sucrose, 20 mM NaC l, 10 mM KC l, 10 mM Tris-C l, pH 7.4, and 10 C i /ml [ 3 H]leucine (121 C i /mmol; IC N Pharmaceuticals, Costa Mesa, CA). Protein concentration was kept at 55 g /ml, within the linear range of the incorporation reaction (Crispino et al, 1993a). One of the two reaction mixtures contained 100 g /ml cycloheximide.…”

Section: Methodsmentioning

confidence: 99%

“…They were used within a day or two of their capture. Optic lobes were dissected from decapitated heads, washed in ice-cold filtered seawater, and processed as described previously (Crispino et al, 1993a). Briefly, a 10% tissue homogenate in 0.7 M sucrose, 20 mM Tris-C l, pH 7.3, was cleared of nuclei and gross particles by centrif ugation in rotor JA-20 of a model J2-21M Beckman centrif uge (3000 rpm, 11 min, 4°C), and the supernatant fraction was centrif uged again in the same rotor at higher speed (12,000 rpm, 30 min, 4°C).…”

Section: Methodsmentioning

confidence: 99%

“…First, the theoretical difficulties and experimental data suggesting that axonal proteins may not be imported exclusively from the perikaryon (Nixon, 1980;Alvarez, 1992) apply even more cogently to the proteins of the presynaptic terminals that are farthest from the nerve cell body and are frequently undergoing long-term plastic changes requiring modifications of their protein complement (Crispino et al, 1993a). Second, the demonstration of local protein synthesis in squid and goldfish axons (see above) makes the hypothesis of the existence of a similar synthetic system in nerve terminals more plausible.…”

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Active Polysomes Are Present in the Large Presynaptic Endings of the Synaptosomal Fraction from Squid Brain

et al. 1997

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Previous data have suggested that the large nerve terminals present in the synaptosomal fraction from squid optic lobe are capable of protein synthesis (Crispino et al., 1993a,b). We have further examined this issue by comparing the translation products of synaptosomal and microsomal polysomes. Both preparations programmed an active process of translation, which was completely abolished by their previous treatment with EDTA. After immunoabsorption of the newly synthesized neurofilament (NF) proteins, the labeling ratio of the 60 and 70 kDa NF proteins was found to differ, in agreement with comparable differences obtained with intact synaptosomes. These observations indicate that the set of mRNAs translated by synaptosomes differs from that translated by nerve cell bodies. Hence, because NF proteins are neuron-specific, they support the view that the active synaptosomal polysomes are mostly localized in the large nerve terminals that represent the most abundant neuronal component of the fraction. This hypothesis was confirmed (1) by electron spectroscopic data demonstrating the presence of ribosomes and polysomes within the large nerve endings of the synaptosomal fraction, as well as in the carrotlike nerve endings of the retinal photoreceptors that constitute the only large terminals in the optic lobe, and (2) by light and high resolution autoradiography of synaptosomal samples incubated with [3 H]leucine, showing that most labeled proteins are associated with the large nerve endings. This response was abolished by cycloheximide. Taken together, the data provide the first unequivocal demonstration that presynaptic nerve terminals are capable of protein synthesis.

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Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions

et al. 1999

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Previous biochemical, autoradiographic, and ultrastructural data have shown that, in the synaptosomal fraction of the squid optic lobe, protein synthesis is largely due to the presynaptic terminals of the retinal photoreceptor neurons (Crispino et al. [1993a] Mol. Cell. Neurosci. 4:366-374; Crispino et al. [1993b] J. Neurochem. 61:1144-1146; Crispino et al. [1997] J. Neurosci. 17:7694-7702). We now report that this process is close to its maximum at the basal concentration of cytosolic Ca++, and is markedly inhibited when the concentration of this ion is either decreased or increased. This conclusion is supported by the results of experiments with: 1) compounds known to increase the level of cytosolic Ca++, such as A23187, ionomycin, thapsigargin, and caffeine; 2) compounds sequestering cytosolic calcium ions such as BAPTA-AM; and 3) agents that block the role of Ca++ as second messenger, such as TFP and W7, which inhibit calmodulin, and calphostin, which inhibits protein kinase C. We conclude that variations in the level of cytosolic Ca++ induced in presynaptic terminals by neuronal activity may contribute to the modulation of the local synthesis of protein.

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Protein Synthesis in a Synaptosomal Fraction from Squid Brain

Cited by 48 publications

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The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons

Active Polysomes Are Present in the Large Presynaptic Endings of the Synaptosomal Fraction from Squid Brain

Protein synthesis in presynaptic endings from squid brain: Modulation by calcium ions

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