Cellular and subcellular localization of protein I in the peripheral nervous system.

Abstract

The cellular and subcellular distribution of protein I, a major brain phosphoprotein, has been studied in the peripheral nervous system. The levels of protein I in various peripheral nerves and innervated peripheral tissues were determined by radioimmunoassay and radioimmunolabeling of polyacrylamide gels. The results indicated that protein I is present throughout the peripheral nervous system. Denervation studies ofadrenal medulla and iris suggested that the protein I contained in peripheral tissues is localized to the neuronal elements innervating those tissues. Protein I was found to be enriched in neurotransmitter vesicle fractions of peripheral nervous tissue. Moreover, protein I appeared to be transported from cell bodies to axon terminals at least partly in association with neurotransmitter vesicles.Protein I is a neuron-specific protein (1) that is a prominent endogenous substrate in brain for both cyclic AMP-dependent (1) and calcium/calmodulin-dependent (2-4) protein kinases. The state of phosphorylation of protein I has been shown to be regulated by impulse conduction (5), neurotransmitters (6, 7), and depolarizing agents (6-8) in intact preparations of neuronal tissue. The distribution of protein I in the central nervous system has been studied by light and electron immunocytochemistry (9, 10), subcellular fractionation (11, t), and radioimmunoassay (12). Protein I is present throughout the central nervous system. It is concentrated in presynaptic nerve terminals, where it is associated at least partially with neurotransmitter vesicles. Protein I has also been detected immunocytochemically, apparently in axon terminals, throughout the peripheral nervous system (13).In the present study we have determined the cellular and subcellular distribution of protein I in various peripheral nervous tissues which have the advantage over brain of being better defined, less complex, and more easily manipulated experimentally. MATERIALS AND METHODSMaterials. Pure protein I was kindly supplied by Louis J.DeGennaro of our laboratory. "2I-Labeled protein A (specific activity, 30 mCi/mg; 1 Ci = 3.7 X 1010 becquerels) was purchased from Amersham and protein A-bearing Staphylococcus aureus cells (SAC) (Pansorbin) were from Calbiochem-Behring.Denervation. Rats with denervated adrenal glands (achieved by transsection of the splanchnic nerve), as well as sham-operated control rats, were obtained from Zivic-Miller Laboratories (Allison Park, PA). Individual adrenal glands were analyzed 2-4 weeks after surgery, at which time denervation is complete (14). Irides were sympathetically denervated by superior cervical ganglionectomy of Sprague-Dawley rats from Anticimex (Stockholm, Sweden). This procedure causes complete degeneration of iris sympathetic nerves within 48 hr (15). Sixteen normal irides and 30 denervated irides, taken 5 days after surgery, were pooled for protein I analysis. Subcellular Fractionation. All of the sucrose solutions used in the fractionation procedures were buffered with 5 mM potassium phosphat...

Synapsin I (protein I), a nerve terminal-specific phosphoprotein. I. Its general distribution in synapses of the central and peripheral nervous system demonstrated by immunofluorescence in frozen and plastic sections.

“…The punctate appearance of synapsin I at these regions suggests that, in axons, too, synapsin I is bound to paniculate structures. The temporal and topographical patterns of accumulation of synapsin I at sites of nerve ligation correlate with the temporal and topographical accumulation of synaptic vesicle-associated antigens (15). This observation is consistent with the possibility that synapsin I migrates down axons in association with synaptic vesicles.…”

“…Similarly, it must be present in the preterminal portion of axons, en route from the cell bodies to the terminals. In fact, experiments involving nerve ligation have demonstrated that, upon local block of axoplasmic transport, synapsin I becomes immunohistochemically detectable (in the form of discrete dots) in the portions of axons close to the ligature (15). The punctate appearance of synapsin I at these regions suggests that, in axons, too, synapsin I is bound to paniculate structures.…”

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve

“…3a). This finding is consistent with previous immunocytochemical studies showing an accumulation of synapsin I proximal to the site of ligation (Fried et al 1982;B66j et al 1986). …”

Influence of spinal cord transection on the presence and axonal transport of CGRP‐, chromogranin A‐, VIP‐, synapsin I‐, and synaptophysin‐like immunoreactivities in rat motor nerve

“…Syn I is localized on intracellular membrane systems and is also affiliated with the exterior surface of synaptic vesicles in its unphosphorylated form. Syn I is rapidly transported with the anterograde axonal transport phase (Fried et al, 1982;, but only a small fraction has been observed to return to the cell body with retrograde transport (Dahlstrom and Booj, 1988;Booj et al, 1989). Synaptophysin (p38) is, however, an integral transmembrane protein (Jahn et al, 1985;Wiedenmann and Franke, 1985).…”