Axon viability and mitochondrial function are dependent on local protein synthesis in sympathetic neurons

Abstract

(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h. Acute inhibition of local protein synthesis also resulted in a significant decrease in the membrane potential of axonal mitochondria. Likewise, blockade of local protein transport into the mitochondria by transfection of the axons with Hsp90 C-terminal domain decreased the mitochondrial membrane potential by 65%. Moreover, inhibition of the local protein synthetic system also reduced the ability of mitochondria to restore axonal levels of ATP after KCl-induced depolarization. (4) Taken together, these results indicate that the local protein synthetic system plays an important role in mitochondrial function and the maintenance of the axon.

Fig. 1

Campenot compartmented cultures. (A) Photo image of a collagen coated 35-mm tissue culture dish with a Teflon chamber attached to the bottom of the dish. The tracks in the collagen covering the area of the lower part of the chamber were made using a pin rake. (B) Schematic drawing of a single track in the bottom of the culture dish showing the location of cell bodies and proximal axons in the center compartment and the growing distal axons in the side compartments. (C) Representative phase contrast photomicrographs of cell bodies in center compartments and axons in side compartments on days 2, 5, and 14 after plating. Images were collected using a Nikon Eclipse TE300 microscope equipped with a KODAK MDS 290 digital camera and saved with Adobe Photoshop 6.0. Bar = 50 μm

Fig. 2

SCG axons contain a heterogeneous population of mRNA. (A and B) RT-PCR analyses performed on total RNA isolated from SCG axons and somas. Note the shift in abundance of β tubulin mRNA relative to H+-ATP synthase and DNA Pol γ mRNAs between axon and soma. Axon, A; Soma, S. (C) COX IV subunit is present in SCG axons, as demonstrated with in situ hybridization. Representative phase contrast photomicrographs of SCG axons after fixation and hybridization with antisense (COX IV antisense) and sense (Control; COX IV sense) riboprobes for the cytochrome c oxidase subunit IV mRNA. Bar = 10 μm

Fig. 3

Protein synthesis inhibitors decrease SCG axon maintenance and translation activity. (A) The attachment of the distal axons in the side compartment was decreased by inhibition of the local protein synthetic system by emetine (100 μM) after 6 h. (B) Inhibition of local protein synthesis by emetine (100 μM) for 15 h resulted in retraction of distal axons toward the center compartment. Representative phase contrast photomicrographs of 14 days-old axons exposed to emetine for 6 h or 15 h and untreated control distal axons in contralateral side compartments. Culture media containing NGF was present in all compartments throughout the duration of the experiment. Arrows indicate regions of axonal detachment and retraction. (C) Axonal maintenance was decreased by inhibition of the local protein synthetic system by emetine, cycloheximide, or chloramphenicol. Representative phase contrast photomicrographs of 14 days-old axons before (0 h) and after 24 h of exposure to emetine (100 μM), cycloheximide (3.5 μM), or chloramphenicol (1 mM). Bars = 50 μm. (D) Axons were pretreated with the protein synthesis inhibitors emetine (10 μM), cycloheximide (350 nM), or chloramphenicol (1 mM) for 30 min and were subsequently incubated for 4 h in methionine-free media containing inhibitors and [³⁵S]Methionine (250 μCi/ml). Axons exposed to culture media containing reagent vehicle lacking inhibitors served as controls. All compartments contained culture media with NGF throughout the experiment. Acid precipitable, alkaline resistant radioactivity was measured on Whatman GFC filter discs by liquid scintillation spectometry as described in methods. Data shown are the mean ± s.e.m. with *P < 0.05 and **P < 0.01 using one sample t-test (two-tailed, α = 0.05, n = 4)

Fig. 4

Inhibition of local protein synthesis decreases axonal mitochondrial membrane potential and generation of ATP. Axons in one side compartment were exposed to the protein synthesis inhibitors emetine (10 μM), cycloheximide (350 nM), or chloramphenicol (1 mM) for 3 h and mitochondrial membrane potential assessed by subsequent treatment with JC-1 at 15.4 μM for 20 min at 37°C (A) or TMRE at 1 μM for 10 min at 37°C (B). Values for mitochondrial membrane potentials, as determined with each dye, were obtained as described in Methods. Data shown are mean ± s.e.m. with *P < 0.05, **P < 0.01, and ***P < 0.01 using one sample t-test (two-tailed, α = 0.05, n = 3–6). (C) Protein synthesis inhibitors impede the recovery of depolarization-induced decreases of axonal ATP levels. Depolarization was induced by exposing untreated SCG axons and axons exposed to emetine or cycloheximide for 3 h followed by a 5 min exposure to 50 mM KCl. Axonal ATP levels were measured using ATPlite 1step kit and a microplate reader. Data shown are mean ± s.e.m. with *P < 0.05 and **P < 0.01 using one sample t-test (two-tailed, α = 0.05, n = 8–9)

Fig. 5

Model of neuronal protein synthesis. Protein synthesis occurs in multiple compartments within neurons to include the dendrite, axon, and nerve terminal. Key features of the model include the rapid and selective transport of stable messenger ribonucleoprotein complexes (mRNPs) to the neuronal periphery and the local regulation of mRNA translation in response to neuronal activity. The model also shows that synthesis of proteins occurs in the vicinity or on the surface of mitochondria in the distal parts of the neuron. The activity in the neuron can be modulated by transcriptional regulation in the soma and translational and post-translational regulation in soma, dendrites, axons, and nerve terminals