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(Hypertension. 2002;39:567.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Obligatory Role of Protein Kinase Cß and MARCKS in Vesicular Trafficking in Living Neurons

Hong Yang; Xiangyu Wang; Colin Sumners; Mohan K. Raizada

Department of Physiology and Functional Genomics, College of Medicine, and University of Florida McKnight Brain Institute, Gainesville, Fla.

Correspondence to Mohan K. Raizada, PhD, Professor, Department of Physiology and Functional Genomics, PO Box 100274, College of Medicine, University of Florida, Gainesville, FL 32610-0274. E-mail mraizada{at}phys.med.ufl.edu

	Abstract

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Neurotransmitter release from neurons involves both vesicular trafficking and subsequent fusion of synaptic vesicles with the plasma membrane. The mechanisms involving the formation and fusion of vesicles that allow the exocytotic release of transmitters are understood well. Little is known, however, about the signaling mechanism involved in the trafficking of vesicles along the neurites. In this study, we used real-time confocal microscopy to search for evidence that vesicular trafficking in neurons requires the activation of protein kinase Cß (PKCß) and the myristoylated alanine-rich C kinase substrate (MARCKS) signaling pathway. Dopamine-ß-hydroxylase fused to green fluorescent protein has been used to trace vesicular movement. Angiotensin II, an established neuromodulatory hormone, stimulates translocation of green fluorescent protein-dopamine-ß-hydroxylase vesicles from the cell body to neurites. This translocation was blocked by an antisense oligonucleotide to PKCß and MARCKS. Stimulation of PKC by other means, such as phorbol-12-myristate-13-acetate or carbachol, also resulted in the redistribution of fluorescence in a manner similar to that observed for angiotensin II. These observations demonstrate that PKCß-MARCKS signaling may be a general mechanism for the stimulation of vesicular trafficking in brain neurons.

Key Words: oligonucleotides, antisense • brain • neuroregulators • norepinephrine • signal transduction

	Introduction

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Both evoked and enhanced neuronal release of transmitters involves trafficking of vesicles along neurites after fusion at the synaptic terminal and the release of exocytosis.^1–2 As a result of extensive investigations during the last 2 decades, the signal transduction mechanism by which synaptic vesicles are formed and subsequently release their transmitters and other contents is fairly well understood.^3–9 Little is known, however, about the signaling mechanism that triggers the trafficking of vesicles from the cell body to the synaptic terminal. Thus, our objective in this investigation was to characterize this signaling system with the use of hypothalamic and brainstem neurons in primary culture and angiotensin II (ang II) as a neuromodulatory hormone. Our rationale for electing to use this experimental system was as follows.

(1) Primary neuronal cultures from the hypothalamus and brainstem areas contain approximately 30% catecholaminergic neurons, and they have been used as an excellent in vitro model to elucidate the regulation of norepinephrine (NE) synthesis, release, and uptake in the brain.^7,10

(2) These neurons provide an excellent electrophysiological and biochemical model with which to study the cellular and molecular basis of physiological actions such as sympathetic activity, baroreflexes, and vasopressin secretion.^7,10–11

(3) Stimulation of the neuronal ang II Type 1 receptor (AT₁R) results in a pattern of NE neuromodulation similar to that seen in the in vivo situation.^12–14

(4) The intracellular signal transduction mechanisms that underlie ang II modulation of NE neurons are fairly well established in this system. For example, the evoked response of ang II on NE neuromodulation involves inhibition of K⁺ channels and stimulation of Ca²⁺ channels via activation of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CAM kinase II).^7,10–11 In contrast, the enhanced response is associated with the transcriptional control of catecholamine synthetic enzymes involving a Ras-Raf-MAP kinase signaling pathway.^{7,10–11,13–14}

(5) The mechanism of AT₁R-induced NE neuromodulation in neurons in vitro is comparable to that observed in vivo.¹²

(6) Our previous studies established that ang II stimulates phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate that is abundant in neurons. MARCKS is involved in the neuromodulatory actions of ang II.¹⁵

Collectively, these observations led us to hypothesize that MARCKS is a key signaling molecule in ang II-induced trafficking of catecholamine vesicles and that its activation is thus an important step in NE neuromodulation. On the basis of this hypothesis, we set out to achieve the following objectives: (1) determine whether ang II causes vesicular translocation/transport, (2) determine whether ang II stimulation of vesicular transport involves a PKCß-MARCKS signaling pathway, and (3) formulate the possible mechanism of this trafficking.

	Methods

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Materials
One-day-old, normotensive Wistar-Kyoto (WKY) rats were obtained from our breeding colony, which originated from Harlan Sprague-Dawley (Indianapolis, Ind). Dulbecco’s modified Eagle medium, plasma-derived horse serum, and x1 crystalline trypsin were purchased from Central Biomedia, Inc. Ang II was purchased from Sigma Chemical Co, and agarose-conjugated protein A/G was obtained from Santa Cruz Biotechnology, Inc. A DßH activity assay kit was purchased from ALPCO. Losartan was a gift from Dupont/Merck, and PD 123319 was obtained from RBI. Monoclonal antibody specific to synaptophysin was obtained from Roche. Antirabbit and antimouse immunoglobulin G, conjugated to HRP, were purchased from Sigma Chemical Co. PCI-green fluorescent protein (pCI-GFP) expression vector was a gift from Professor Gerry Shaw, Department of Neuroscience, College of Medicine, University of Florida, McKnight Brain Institute (Gainesville, Fla). All other reagents were purchased from Fisher Scientific. Sense oligonucleotides (SONs) and antisense oligonucleotides (AONs) specific for MARCKS and PKC subtypes were synthesized by the Interdisciplinary Center for Biotechnology Research, University of Florida, whose sequences and the effectiveness thereof we described previously.¹⁵

Procedures
Neuronal Cell in Primary Culture From Rat Brain Culture
Neuronal cultures were prepared essentially as described previously.^16–17 In brief, the hypothalamus and brainstem areas of 1-day-old WKY rat brains were dissected, and brain cells were dissociated with trypsin. Cells were placed onto poly-L-lysine-precoated cover glass chambers (Nalge Nunc) in Dulbecco’s modified Eagle medium containing 10% plasma-derived horse serum essentially as described previously.^16–17 The cultures that contained more than 90% neurons and remaining astroglia were maintained at 37°C in 10% CO₂:90% O₂ before DNA transfection.

Plasmids
The cDNA-encoding full-length coding region of rat dopamine-ß-hydroxylase (DßH) with GFP was cloned in mammalian expression vector pCI-Neo under the control of the cytomegalovirus promoter, as described previously.¹⁸ The construct was verified by restriction analysis and DNA sequencing.

Transfection of Neurons With pCI-GFP-DßH Plasmid
Hypothalamus-brainstem neurons from WKY rat brain were transfected by pCI-GFP-DßH plasmid using a modified calcium phosphate transfection protocol, essentially as described previously.^18,19 In control experiments, neurons were transfected with pCI-GFP plasmid.

Confocal Microscopic Analysis of GFP-DßH Fluorescent Images of Neuronal Cells
Neuronal cultures were grown on chambered cover glass. After transfection, GFP-DßH expression was detected and photographed with a 1024ES Confocal Scanning Laser Microscope (Bio-Rad) using a Fluotar 100x/1.3 objective, as described previously.¹⁸ The images were captured and explored as tagged image file format (TIFF) files and analyzed with Scion Image software (Scion Co). To determine relative intensity, data were quantified using a line intensity profile across the neurites. For each set of conditions, the intensity of the pixels was summed within the individual neurites of at least four neurons, essentially as described previously.²⁰ The results were normalized, with the value at 0 minutes being 1.

	Results

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A cDNA encoding the full-length coding region of the rat DßH-GFP was cloned in a mammalian expression vector (pCI-Neo) under the control of cytomegalovirus promoters. We decided to use GFP-DßH for vesicular trafficking because DßH is a well-established marker for neuronal vesicles.^21–22 The transfection efficiency of the plasmid in culture cells and the expression of DßH activity of the GFP-DßH fusion protein were established in previous studies.¹⁸ For example, transfection of TE 671 cells with the pCI-GFP-DßH plasmid resulted in the expression of fluorescence in 80 to 90% of the cells within 24 hours and was associated with a tenfold increase in DßH enzyme activity in pCI-GFP-DßH as compared with the control (pCI-GFP) cells. A transfection protocol has been developed for neurons that yields approximately 2% transfection efficiency with excellent morphological preservation of neurons in primary cell culture. Neurons depict bright punctuate fluorescence representing GFP-DßH that is predominantly localized in the cell soma as well as in some of the neurites.

The effect of 100 nmol/L ang II on the distribution of GFP-DßH was studied in living neurons to confirm our previous studies that this hormone induces redistribution of vesicles using real-time confocal microscopy.¹⁸ Few neurites display GFP-DßH at zero time. In the presence of ang II, however, both the intensity and the number of neurites depicting fluorescence was significantly increased. The redistribution of GFP-DßH was rapid, becoming apparent within 1 minute and persisting throughout 30 minutes. Under control conditions only 2±0.5 (n=20), neurites exhibited a faint fluorescence. The number of GFP-DßH positive neurites increased to 4.5±0.4 (n=20) when neurons were incubated with 100 nmol/L ang II. In contrast to GFP-DßH-transfected neurons, ang II had no effect on fluorescent distribution in control neurons that were transfected with GFP construct alone (Figure 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Photomicrographs showing the effect of ang II on distribution of GFP in brain neurons. Neurons were transfected with pCI-GFP plasmid as described in Methods. Forty-eight hours after transfection, cells were incubated with 100 nmol/L ang II for 30 minutes. A 1024 ES Confocal Scanning Laser microscope (Bio-Rad) was used to record the distribution of fluorescence. The images presented are representative of 10 neurons. Bar represents 15 µm.

Results of previous studies indicate that AT₁R stimulation activates Ca²⁺-dependent PKC.^7,10 Thus, our next objective was to determine the involvement of this enzyme by studying the effect of the PKC activator phorbol-12-myristate-13-acetate (PMA) on the redistribution of GFP-DßH. Incubation with 100 nmol/L PMA, which elicits a two- to fourfold increase in neuronal PKC activity, resulted in a dramatic redistribution of GFP-DßH in the neurites within 15 minutes (Figure 2). The pattern of distribution was similar to that observed with ang II treatment.¹⁸ Longer preincubation with PMA (24 h), which causes downregulation of neuronal PKC,²³ had little effect on GFP-DßH redistribution to the neurites. These observations, taken together with the ang II data, indicate that activation of PKC is the key in vesicular trafficking. Neuronal cultures were treated with carbachol for 15 minutes to determine whether stimulation of PKC by other agonists would also induce vesicular trafficking. Carbachol seemed to be an appropriate choice because our previous data showed that neurons express muscarinic receptors and that their activation by carbachol results in the activation of IP₃-PKC signaling pathway.^12,24 Treatment of neurons with carbachol (100 µmol/L) caused distribution of GFP-DßH along neurites in a manner similar to that observed with ang II. Figure 3 is a representative image after examination of multiple live neurons. Examination of neurites for 10 minutes before 100 µmol/L carbachol was used to observe the movement of fluorescent vesicles independently of carbachol (Figure 3a). A broken vertical line was used as a guide to track the movement of clusters. Treatment with carbachol caused four clusters (pink, yellow, blue, and red) to move. The movement of each cluster appeared to be distinct (Figure 3b to 3d).

View larger version (26K): [in this window] [in a new window]   Figure 2. Photomicrographs showing the effect of PMA on GFP-DßH distribution in brain neurons. Neurons transfected with pCI-GFP-DßH were incubated with 100 nmol/L PMA. Distribution of fluorescence was examined as a function of time. Images from a single neuron are representative of 20 neurons examined. Bar represents 15 µm.

View larger version (55K): [in this window] [in a new window]   Figure 3. Photomicrographs showing the effect of carbachol on a real-time movement of fluorescent clusters in a neurite. A distal area of neurite of GFP-DßH transfected neuron was examined for 10 minutes before carbachol treatment (a) to identify the movement of fluorescent clusters independently of carbachol. Carbachol 100 µmol/L was added at zero time (b), and movement of GFP-DßH was captured at 0, 10, and 20 minutes (c, d). The vertical dotted line is a landmark to show the relative movement of vesicles. Data presented are representative of 10 neurites from 2 independent experiments.

Ca²⁺-dependent PKC subtypes were depleted with the use of homologous AONs to PKC, PKCß, and PKC. In previous studies, we established that treatment of neurons with 2 µmol/L AON but not the corresponding SONs for 24 to 48 hours results in an 80 to 85% decrease in the homologous PKC subtypes.¹⁵ This depletion was highly selective because heterologous AON showed no effect.¹⁵ For example, treatment of neurons with PKCß AON selectively decreases endogenous levels of PKCß without any effect on PKC or PKC. Pretreatment of neurons with PKCß subtype AON resulted in a significant decrease in the GFP-DßH distribution in the unstimulated state. In addition, ang II-induced GFP-DßH distribution to the neurites was attenuated (Figure 4). A majority of the fluorescence remained in the neuronal cell soma, which indicates that 80 to 85% depletion of PKCß results in a significant inhibition of translocation of fluorescence to the neurites. This conclusion was specific and PKCß-dependent and was further supported by the fact that AONs to PKC and PKC had no effect on ang II-induced redistribution (Figure 4). The conditions that depleted PKCß and attenuated ang II-induced GFP-DßH distribution had no effect on basal vesicular formation. This conclusion was made on the basis of the observation that levels of markers of vesicles such as synaptophysin and DßH were not altered by PKCß AON depletion (Figure 5).

View larger version (65K): [in this window] [in a new window]   Figure 4. Photomicrographs showing the effects of PKC, PKCß, and PKC depletion on ang II-induced GFP-DßH distribution on brain neurons. Neuronal cultures were co-transfected with AONs or SONs of PKC, PKCß, or PKC with pCI-GFP-DßH sequence for the specific AON and SON and experimental conditions published.15 This treatment results in an 80% decrease in PKC subtype homologous to specific AON in 48 hours. Cultures were treated with 100 nmol/L ang II after AONs treatment, and live neurons were examined for the distribution of green fluorescence. Images are representative of 25 to 30 neurons. Bar represents 15 µm.

View larger version (39K): [in this window] [in a new window]   Figure 5. Bar graph illustrating the effect of PKCß depletion on synaptophysin and DßH immunoreactivities. Neuronal cultures were transfected with AON or SON to PKCß.15 Cell lysates were prepared and levels of synaptophysin and DßH immunoreactivities were determined, as described elsewhere15 and in Methods. The data represent the means of 2 experiments.

We also studied the role of MARCKS in ang II-PKCß-mediated stimulation of GFP-DßH distribution. The rationale for selecting MARCKS as a possible target was based on our previous findings,¹⁵ which indicate that ang II stimulates phosphorylation of MARCKS and its redistribution in neurons, an effect that is mediated by the PKCß subtype. In addition, MARCKS phosphorylation and redistribution are key in ang II stimulation of NE neuromodulation. Endogenous MARCKS was depleted by preincubation of neurons with 2 µm AON specific to MARCKS for 48 hours at 37°C. This treatment has been established to cause 77% depletion of endogenous MARCKS immunoreactivity and a parallel decrease in ang II-induced phosphorylated MARCKS.¹⁵ The effect of AONs was specific because SONs to MARCKS had no effect. Pretreatment of neuronal cultures with MARCKS-AON resulted in the failure of ang II to stimulate distribution of GFP-DßH into the neurites (Figure 6). To further confirm the involvement of the PKCß-MARCKS pathway, neuronal cultures were subjected to PKCß or MARCKS depletion by their respective AONs, and the effect of PMA on GFP-DßH redistribution was examined. In both cases, PMA failed to induce the translocation of fluorescence into the neurites (Figure 7). In neurons that were treated with PKCß or MARCKS SONS, PMA exhibited redistribution of fluorescence in the neurites, which was similar to that seen in ang II-treated control neurons.

View larger version (34K): [in this window] [in a new window]   Figure 6. Photomicrographs showing the effect of depletion of MARCKS on ang II-induced distribution of GFP-DßH in neurons. Neuronal cultures were co-transfected with MARCKS-SON (5'-AAGAAGCCAGCATGGGTGCACACTT-3') or MARCKS-AON (5'-AACTGTGCACCCATGCTGGCTTGTT-3') and pCI-GFP-DßH for 48 hours. This was followed by an examination of green fluorescence in live neurons after treatment with 100 nmol/L ang II for 15 minutes. Images represent 35 neurons. Bar represents 15 µm.

View larger version (70K): [in this window] [in a new window]   Figure 7. Photomicrographs showing the effect of depletion of PKCß and MARCKS on PMA-induced distribution of GFP-DßH in neurons. Neuronal cultures were co-transfected with AONs of PKCß or MARCKS and pCI-GFP-DßH for 48 hours. PMA 100 nmol/L was added to neuronal cultures after AON treatment, and live neurons were examined for the distribution of green fluorescence. Images represent 20 neurons. Bar represents 15 µmol/L.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This study proves that PKCß-MARCKS serves as an obligatory signaling pathway in the trafficking of vesicles containing DßH from the cell body to the neurites. The evidence that we found includes the following: (1) ang II stimulates Ca²⁺-dependent PKC, and depletion of PKCß alone enable neurons to be nonresponsive to ang II;¹⁵ (2) ang II causes phosphorylation of MARCKS, which is key in the vesicular trafficking and NE neuromodulation (this study and that of Lu et al¹⁵); and (3) depletion of PKCß or MARCKS individually resulted in the inability of PMA to stimulate vesicular trafficking. The effect of ang II seems to be on the trafficking of vesicles and not on the formation and/or synthesis of vesicles. This conclusion is supported by the observations that (1) inhibition of vesicle formation by actinomycin D had no effect on vesicular trafficking and (2) depletion of PKCß has little effect on cellular levels of vesicular proteins, synaptophysin, and DßH (this study).

An important question that arises from this study concerns the mechanism by which MARCKS facilitates and/or stimulates vesicular trafficking. MARCKS is a member of small family of proteins that bind calmodulin in the presence of calcium and also bind and cross-link actin in a mutually exclusive fashion. This fact, coupled with observations that translocation and trafficking of vesicles and axonal transport of organelles involves microtubules and is facilitated by flexibility of actin filaments, leads us to propose that MARCKS plays a key role in the regulation of these two cytoskeletal elements. It is tempting to speculate that vesicles are bound to cytoskeletal elements and other dephosphorylated proteins, which keeps the vesicles in a state of reserve. Stimulation of PKCß activates MARCKS and releases calmodulin. Calmodulin activates CAM kinase II, which phosphorylates cytoskeletal, bound proteins, thus releasing the vesicles from the reserve to the active state. This process allows trafficking and exocytosis to occur. Sufficient evidence exists to support this hypothesis: (1) ang II stimulates calmodulin-CAM kinase II activity in neurons;²⁵ (2) the calmodulin-CAM kinase II pathway is known to phosphorylate one such protein involved in exocytosis, synapsin I;^26–27 (3) W-7 (100 µmol/L), an inhibitor of calmodulin, attenuates ang II-induced vesicular trafficking to the neurites (data not shown). The possibility that MARCKS-calmodulin may regulate cytoskeletal elements directly to regulate trafficking cannot be ruled out. Nonetheless, this study provides strong evidence for a PKCß-MARCKS-calmodulin-Cam kinase II signaling pathway in vesicular trafficking in catecholaminergic neurons.

	Acknowledgments

 
This work was supported by grant HL33610 to M.K.R. from the National Institutes of Health. We thank Marya Fancey and Mary Spivey for their editorial support and Ling Liu for technical support. Dr Wang was a visiting fellow in the Division of Cardiology and Hypertension, Fujian Medical University, Fuzhou, People’s Republic of China.

Received September 23, 2001; first decision October 25, 2001; accepted November 6, 2001.

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