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,2,2
* Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden;
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York, USA;
Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, USA;
Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, Kentucky, USA; and
|| Department of Morphological Brain Science, Kyoto University, Kyoto, Japan
3Correspondence: Experimental Alcohol and Drug Addiction Research Section, CMM L8:01, Department of Clinical Neuroscience, Karolinska Institute, S-171 76 Stockholm, Sweden. E-mail: georgy.bakalkin{at}ki.se
SPECIFIC AIMS
The classical model postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network and in secretory vesicles during axonal transport (Fig. 1
A). To test this model, we have characterized prodynorphin (PDYN), precursor protein to the dynorphin opioid peptides; identified cellular distribution and subcellular sites in neurons in which this protein is stored and processed to mature peptides; and evaluated whether stimuli that induce dynorphin secretion also stimulate PDYN processing.
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PRINCIPAL FINDINGS
1. Characterization of PDYN in rat brain
PDYN levels and distribution in the brain were characterized by WB and immunocytochemistry (IC) with Affinity-purified anti-rat PDYN-antibodies directed against the C-terminal 235–248 fragment. PDYN was detected in several brain areas, including the hippocampus, striatum, and cerebral cortex by WB as a dominant band with an apparent molecular size of 28 kDa identical to the calculated size. This band was absent in PDYN knockout mice. PDYN processing intermediates were not evident in brain tissues, suggesting that PDYN is either rapidly processed to opioid peptides or is stored in an unprocessed form.
Analysis of the biochemical properties revealed a low solubility of PDYN. Insoluble PDYN may consist of oligomers stabilized by intermolecular disulphide bonds formed by six Cys residues, remaining at the PDYN N terminus after cleavage of the signal peptide. Consistently, high molecular weight PDYN complexes were identified by PAGE under nonreducing conditions. These complexes dissociated to PDYN monomers in the presence of the reducing agent DTT.
2. Regional variation in PDYN distribution patterns
Light microscopic examination demonstrated a characteristic distribution pattern of PDYN immunoreactivity (PDYN-IR) in the brain. This pattern was generally consistent with previous analyses of dynorphin peptides with high levels in the hippocampus, striatum, amygdala, and ventral tegmental area (VTA) and lower amounts in the cerebral cortex. Hippocampus displayed PDYN-IR in the hilus and stratum lacunolum-moleculare of the CA3 region. In the amygdala, neurons displayed intense IR perikarya and dendrites. Neurons in the cerebral cortex displayed PDYN-IR mostly in cell bodies.
The PDYN-IR pattern was nearly identical to that revealed for Dyn A. The presence of PDYN-IR in brain structures such as the CA3 region in the hippocampus and VTA, which lack PDYN-containing neuronal cell bodies, suggests that PDYN is transported to and stored in axon terminals (Fig. 1B
).
3. Ultrastructural localization of PDYN and dynorphin peptides
Electron microscopic IC showed PDYN-IR in many somatodendritic profiles in rat hippocampal dentate gyrus and CA3 areas and in the nucleus accumbens shell. The hippocampal PDYN-IR somata were identified as granule cells. Within these cells, PDYN-IR was primarily associated with vesicular organelles resembling dense core vesicles. Occasionally, the PDYN-labeled granules were affiliated with the Golgi apparatus. PDYN-IR was exclusively seen in small axons and axon terminals in the hilus and supragranular regions of the dentate gyrus and in stratum lucidum of CA3 (Fig. 2
). These terminals contained one to five dense core vesicles and numerous small synaptic vesicles. Within the hilus and CA3, the majority of PDYN-labeled axonal profiles were mossy fiber terminals, since they were large (>1.0 µm in diameter) and formed synapses with multiple dendritic spines as well as dendritic shafts. In dual- labeling experiments, the PDYN was shown to coexist with dynorphin A in the same axon terminal, where there was occasional codistribution in individual dense core vesicles.
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4. PDYN/dynorphin peptide ratio in axon terminals
We next evaluated whether PDYN is present in the CA3 area and VTA in unprocessed or processed forms and assessed the proportion of an unprocessed PDYN compared with mature peptides in these structures (Fig. 3
). PDYN and dynorphin content was compared using the combination of WB and RIA after trypsin digestion to Leu-enkephalin-Arg, a PDYN marker. PDYN was partially purified from rat pituitary tissue by PAGE and extracted from the gel, and protein aliquots were used as a standard in WB experiments. Molar content of PDYN in a preparation was determined by protein digestion with trypsin that liberates Leu-enkephalin-Arg, which was separated by SP-Sephadex C-25 chromatography and measured by RIA.
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The molar content of PDYN substantially exceeded endogenous levels of Dyn A, Dyn B, and Leu-enkephalin-Arg by 2- to 10-fold in structures where PDYN is synthesized (the dentate gyrus and striatum) and in areas containing axon terminals of PDYN-containing neurons (the CA3 area and VTA; Fig. 3
).
5. Regulation of PDYN processing
PDYN processing and dynorphin secretion in axon terminals may be regulated in concert or differentially. The 90 min stimulation of neuronal activity by kainic acid (10 mg/kg ip), too short-lasting to cause neuronal death in the hippocampus, decreased the levels of Dyn A, Dyn B, and Leu-enkephalin-Arg in CA3 region approximately by half, whereas the PDYN content remained unchanged (Fig. 3C
). These decreases may occur due to stimulation of release or degradation of opioid peptides. Regardless of the mechanism, these data demonstrate that PDYN processing and dynorphin secretion are differentially regulated in axon terminals.
PDYN processing in axon terminals and dendrites may be regulated by cell depolarization that also induces the release of mature neuropeptides. Incubation of embryonic cortical neurons for 2 h in the presence of depolarizing potassium concentrations stimulated the release of Dyn B and Leu-enkephalin-Arg into the medium by 3.5- to 6-fold, whereas peptide levels in the cells were not increased. The total levels of PDYN-derived peptides in the medium T cells were increased by 3-fold, demonstrating that PDYN processing was activated by depolarization.
CONCLUSIONS AND SIGNIFICANCE
Neuropeptide precursors have long been thought to be processed into short functional fragments in secretory granules during transport from neuronal soma to axon terminals. In the present study, we provide pivotal evidence that PDYN is present in axon terminals along with dynorphin opioid peptides. We also show that in the CA3 and VTA regions containing PDYN terminals arising from the dentate gyrus and striatum, respectively, the molar content of PDYN is much greater than that of dynorphins. We hypothesize that PDYN storage and processing at synapses may provide a local mechanism for regulation of synaptic transmission (Fig. 1B
).
PDYN-IR was also observed in dense granular aggregates in dendrites as well as somata of dentate granule cells and striatal neurons, where ultrastructural dual labeling shows coexistence with dynorphin peptides. These results are consistent with prior evidence that neuropeptides are present in large dense core vesicles in the dendritic tree of many neurons. Dendritic secretion of dynorphins, oxytocin, and vasopressin appears to be an important mechanism in the local regulation of synaptic transmission. These data along with the presence of PDYN in dendrites in the hippocampus, cerebral cortex, and amygdala suggest that targeting of neuropeptide precursors to dendrites and their storage and processing in this compartment represents a general mechanism in the brain.
A fraction of PDYN is apparently present in brain structures in an insoluble form that may include oligomers stabilized by disulfide bridges. The disulfide bonds are essential for the assembly and oligomerization of secretory proteins that determine their intracellular transport. PDYN oligomers may also represent the storage form of the protein. A substantial amplification of the signal is possible if PDYN, stored in a condensed form, is processed shortly prior or during release of mature opioid peptides.
Depolarization-induced activation of PDYN processing was observed in neuronal cultures. This may occur due to activation of either biosynthesis or activity of the PC2 processing enzyme or decondensation of the granule content in response to depolarization. With respect to the association of processing and secretion, PDYN may represent a similar case as proatrial natriuretic factor, which undergoes cleavage and secretion concomitantly when activated by extracellular stimuli. Storage of PDYN and stimulation of its processing in axon terminals and dendrites by neuronal activity or extracellular signals may represent a mechanism for the local regulation of synaptic transmission.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6174fje
1 These authors contributed equally to this study. 
2 These authors contributed equally to this study.
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