Dophin 2008 Article in the Journal Neuroscience & Biobehavioral Review Sleep
How Postdoctoral Research in Paul Greengard'southward Laboratory Shaped My Scientific Career, Although I Never Did Another Phosphorylation Assay
Abstract
In this short review, I describe from personal experience how every step in the career of any scientist, no matter how disjointed and businesslike each might seem at the time, will nigh inevitably meld together, to assistance united states all tackle novel projects. My postdoctoral research in Paul Greengard's laboratory, where I investigated neurotransmitter-mediated phosphorylation of Synapsin I, was instrumental in my career progression, and Paul's support was instrumental in my power to make a spring into independent research.
When the Society for Neuroscience was soliciting articles for its new journal, I was a postdoctoral fellow in Paul Greengard'southward laboratory, completing studies of neurotransmitter regulation of synapsin I phosphorylation. When Paul informed me that he been invited to contribute a newspaper, nosotros knew it would exist risky affair to volunteer one's precious information because this was an untested journal. But it came at a good time because I was about to leave the laboratory and had data that complemented my recently accustomed paper demonstrating phosphorylation of the presynaptic protein Synapsin I (which we chosen Protein I at the time) by a neurotransmitter (Dolphin and Greengard, 1981b). So I was very happy to publish in the initial volume of the Journal of Neuroscience, which was to be the outset of my 17 papers then far in the journal. The information published in 1981 extended my work on serotonin-mediated phosphorylation and as well demonstrated phosphorylation by another course of neuromodulator, acting at adenosine receptors (Dolphin and Greengard, 1981a). It was such a pleasure for me to reread that newspaper, equally information technology brought back many memories of life in the Greengard laboratory, together with colleagues and friends, including Pietro De Camilli, Angus Nairn, Wieland Huttner, Clive Palfrey, Eric Nestler, Suzanne Lohmann, Ulrich Walter, Howard Schulman, Mary Kennedy, and many others.
My interest in intracellular signaling pathways activated by neurotransmitters began when I was an undergraduate student in Biochemistry; I wrote a dissertation on cyclic AMP in prokaryotes and eukaryotes, and then conducted an undergraduate enquiry projection examining glycine as a neurotransmitter, using spinal cord synaptosomes equally an experimental preparation. This interest propelled me toward Parkinson's disease research every bit a graduate student in the laboratory of the neurologist David Marsden at the Establish of Psychiatry in London, where I examined the importance of dopaminergic and noradrenergic pathways in animal models of Parkinson's disease. I so wrote to Paul Greengard, asking to practice postdoctoral enquiry nether his mentorship, because I knew I needed more bones preparation in cell signaling pathways, since my PhD environment had been very focused on translational and clinical research. I could not think of anywhere improve than Paul's laboratory because of his seminal work on cyclic nucleotide signaling pathways (Axle and Greengard, 1976). He said his laboratory was total, merely agreed to have me in a year'south time, subject to interview. Fortunately, I had also obtained a UK Medical Inquiry Council French exchange fellowship to piece of work in the laboratory of Joel Bockaert, at College de France in Paris, where I studied the coupling between β-adrenergic receptors and adenylate cyclase. This valuable feel cemented my desire to go along research in this area and work with Paul Greengard. Thus it was that Paul, on a visit to Paris, invited me to exist interviewed in his hotel room. I was on my baby-sit when I knocked on the door, and a vocalism called out to come in. Equally I entered, I could run across nothing just a large bed, simply I soon located Paul lying flat on the floor across; he explained information technology was because of his chronic back problem. I perched on the bed, and tried to answer his searching scientific questions. I later realized that Paul lay on the flooring at every opportunity, oft in our laboratory meetings and indeed during other interviews (Nestler, 2019).
When I arrived in Paul'due south laboratory and so at Yale, in the summertime of 1978, the protein Synapsin I had recently been identified as a major presynaptic phosphoprotein (Ueda and Greengard, 1977). It was and then called "Protein 1" because it stood out equally the primary polypeptide in synaptosomes that was phosphorylated in both a cAMP- and a Ca2+-dependent manner (Sieghart et al., 1979). However, no neurotransmitters had yet been found to influence the phosphorylation of Synapsin I, and to identify such a pathway seemed to exist the holy grail at the fourth dimension. Paul's world view of multiple signaling cascades terminating in the phosphorylation of many different target proteins, that would underlie long-term synaptic events, was frequently at odds with the then prevailing view of ion aqueduct biologists, that neurotransmission was mediated by fast neurotransmitters acting on ion channels, a process in which at that place was no need to consider neurochemical mechanisms. Eric Kandel (from an electrophysiological standpoint), and Paul, from the basis of biochemical pathways, were both key to the acceptance of the view that a multitude of biochemical pathways must play a role in longer term events. Indeed, Paul was collaborating with Eric Kandel'due south laboratory at that time to elucidate whether a phosphorylation pour mediated the effects of serotonin (five-HT) in Aplysia, and many of us in the laboratory were very influenced by these elegant studies, combining, equally they did, electrophysiology and biochemical approaches (Castellucci et al., 1980, 1982).
Paul suggested that my project should be to examine whether neurotransmitters could change the state of phosphorylation of Synapsin I in the encephalon, only he left information technology entirely to me what tissue I should utilize for my study. There were ∼25 postdocs and students in his laboratory at the time, and then I had a lot of aid and advice, although Paul was the opposite of a micro-manager. Influenced both past the work of Eric Kandel and that of my previous mentor Joel Bockaert (Enjalbert et al., 1978), I decided that serotonin would be a good neurotransmitter to study. Taking inspiration from the work of George Aghajanian, also a professor in the Pharmacology Section at Yale, whose studies had revealed the effects of serotonin on many types of neuron (Aghajanian et al., 1990), I chose to examine the tiny facial nucleus in the brainstem, since Aghajanian had extensively examined the electrophysiological backdrop of this nucleus, equally well as its ultrastructure (Aghajanian and McCall, 1980). His work indicated the facial nucleus had no interneurons, and therefore I idea it might lead to a more homogeneous phosphorylation response than many other brain areas. Indeed, serotonin did upshot in an increase in phosphorylation of Synapsin I in this preparation, merely I initially found that the furnishings were modest and very variable (Fig. 1, left). This was disappointing, and I recall blaming myself for being a cack-handed experimenter.
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Figure ane.
Phosphorylation of Synapsin I by deportment at serotonin and adenosine receptors. Stimulation of Synapsin I (then called Protein I) phosphorylation with serotonin in the presence and absenteeism of IBMX (left) (from Dolphin and Greengard, 1981b, our Fig. 2) or 2-choloroadenosine (correct) (from Dolphin and Greengard, 1981a, our Fig. 4). The "back phosphorylation" method was used, in which 32P-ATP together with catalytic subunit of protein kinase A were used to phosphorylate residuum nonphosphorylated sites on Synapsin I.
A quantum came indirectly through family tragedy; my father died in 1979, and I returned to England for the funeral. Fortuitously, I besides decided to investigate potential future positions in the United kingdom, and visited Leslie Iversen'due south Medical Enquiry Council unit in Cambridge. While in that location, I showed my underwhelming phosphorylation data to John Dowling, who was on sabbatical in the unit of measurement at the time, working on dopamine-sensitive adenylate cyclase activity in the retina (Watling et al., 1979). I asked his advice and remember clearly that he suggested calculation a low concentration of a phosphodiesterase inhibitor to the slices to potentiate the cAMP response to serotonin. On my return, I duly added isobutyl-methyl-xanthine (IBMX) to the facial nucleus slices, and the responses became much more than robust (Dolphin and Greengard, 1981b) (Fig. 1, left). Similarly, Eric Nestler besides found that dopamine and depolarization increased the phosphorylation of Synapsin I in superior cervical ganglion neurons (Nestler and Greengard, 1980).
Something I could not understand in my data was that the serotonin-mediated phosphorylation of Synapsin I was Catwo+-dependent, whereas the phosphorylation mediated past stimulation of an adenosine receptor was not. This was the main topic of my follow-upwardly paper in the first volume of Journal of Neuroscience (Dolphin and Greengard, 1981a) (Fig. 1, right). Everyone in the laboratory remembers that writing papers with Paul was exhausting, as we considered multiple versions of every judgement, and these were written and rewritten in pencil with many erasures. But I can still hear his voice in every phrase of this Journal of Neuroscience paper, and it remains a pleasure to reread each well-considered argument, for which I have admittedly no credit.
In the Discussion, many possibilities are suggested to explain the surprising Ca2+ dependence of the serotonin-stimulated phosphorylation, and these points remain relevant, even now. For example, we indicate out that, in Aplysia neurons, serotonin stimulated a presynaptic adenylate cyclase, and this increased the influx of Ca2+ (Shimahara and Tauc, 1977; Klein and Kandel, 1978). Nosotros suggested that both processes might contribute to presynaptic Synapsin I phosphorylation. I likewise wonder now if we were seeing a combined effect of 5HT3 receptor activation, allowing Ca2+ entry, and subsequent Ca2+-calmodulin (CaM) kinase-dependent phosphorylation of Synapsin I, also equally activation of presynaptic serotonergic GPCRs that direct stimulated adenylate cyclase. Of grade, the existence of 5HT3 receptors, which are Catwo+-permeable ion channels, and the many subtypes of G-protein-coupled 5-HT receptors was unknown at the time (Raymond et al., 2001).
My study of neurotransmitter-dependent phosphorylation of Synapsin I was cut brusque because of family unit issues that called me back to the Uk. But work on Synapsin I simultaneously involved many others in Paul'southward laboratory, and so research on its subcellular localization and function continued quickly. For instance, Wieland Huttner and Mary Kennedy both contributed to unraveling the procedure of Ca2+-dependent phosphorylation of Synapsin I (Huttner et al., 1981; Kennedy and Greengard, 1981). Indeed, cAMP-dependent poly peptide kinase and CaM kinase both phosphorylated the same site on Synapsin I, and CaM kinase also phosphorylated boosted sites (Huttner et al., 1981). Pietro De Camilli showed in elegant immuno-electron microscopic studies that Synapsin I was a cytoplasmic protein that was associated with synaptic vesicles (De Camilli et al., 1983). To date, there are over 2000 studies involving Synapsin I listed in PubMed, identifying its roles in synaptic function and pathology. These include many from Paul's own laboratory, standing throughout his career (Hilfiker et al., 2005; Venton et al., 2006).
When I returned to UK, I had the adept fortune to obtain a staff position at the Medical Enquiry Council's National Establish of Medical Research (NIMR) at Manufacturing plant Hill in London. This was an institution that Paul knew well, since he had spent a postdoctoral period in Wilhelm Feldberg'south department at NIMR in the 1950s, recording from sympathetic nerve fibers. I find it fascinating that Paul was already interested in phosphorylation even and then (Greengard and Straub, 1959a, 1959b). My post came about entirely through Paul's suggestion and generosity: he wrote to Arnold Burgen, and then the Director of the Establish, whom he must have known from his work on cholinergic manual. All of a sudden and miraculously, I was offered a chore, during a flow when Margaret Thatcher was Prime Government minister, the UK was in a deep recession, and academic positions were few and far betwixt.
Before arriving, I knew very little about NIMR, and was thrilled to be appointed into the same Partitioning of Neurophysiology and Neuropharmacology, which had previously housed the Yale triumvirate of Paul Greengard, Murdoch Ritchie, and Bill Douglas, and which currently contained an eclectic and heady mix of groups studying pain, hearing, epilepsy, and hippocampal LTP. Wilhelm Feldberg also still had a laboratory there, and used to phone call me Delphine. I was, all the same, supernumerary, and given a small cupboard to work in, and an fifty-fifty smaller research consumables budget. In that surroundings, since I had go intrigued past presynaptic events, I decided to examine neurotransmitter release, and its modulation by activation of presynaptic receptors, including those activated by adenosine agonists, edifice on my knowledge of the very robust response of Synapsin I phosphorylation (Dolphin, 1983; Dolphin and Archer, 1983). I also rapidly developed an enjoyable collaboration with Tim Bliss and members of his group, including Mick Errington, working with them to measure out field potentials and examine glutamate release from the dentate gyrus during the induction of LTP (Dolphin et al., 1982).
Studying the regulation of neurotransmitter release naturally led me to further questions most regulation of the Ca2+ influx that triggers this release, but this was not something I could directly address at NIMR. Although information technology did not feel like it at the time, it was fortuitous that the rules for obtaining tenure changed while I was at that place, meaning that to apply for tenure I would have to reapply for my position and then remain untenured for another five years. Many of the affected staff started to look for university posts, and I was offered a lectureship at St. George's Hospital Medical School, in the Department of Pharmacology. The interview went well, autonomously from being asked if I intended to accept children, and I moved there in 1983, hoping once more to pivot my research in a new direction. At that fourth dimension, the Department was almost entirely populated by electrophysiologists, including the Head of Department, John Kelly, together with Vincenzo Crunelli, Mark Mayer, and Tom Bolton.
In this environs, it would be feasible for me to written report voltage-gated calcium channels and their modulation by neurotransmitters, and how this related to neurotransmitter release. Ironically, while I was at Yale, Richard Tsien was on the faculty in the Physiology department, doing beautiful work on cardiac electrophysiology, leading upwardly to an understanding of how cAMP modulates cardiac calcium channels, and the existence of additional calcium channels in neurons (Bean et al., 1984; Nowycky et al., 1985). Yet, my main retention of Dick Tsien at the time was that he adjudicated on my Yale postdoctoral fellowship application. When I started investigating calcium channels, I very belatedly wished I had concentrated better during Physiology seminars at Yale, to take advantage of their wealth of cognition. Nevertheless, through the enormous generosity specially of John Kelly at St. George's, I was able to learn how to record calcium currents in DRG neurons, which had already been established as a model to study presynaptic events (Dunlap and Fischbach, 1978). Together with my first postdoctoral research assistant and great friend, Rod Scott, we started to apply the same techniques to examine the mechanism of modulation of action potentials and calcium currents in these neurons by neurotransmitters, where we showed inhibition, particularly by activation of GABA-B and adenosine receptors (Dolphin et al., 1986).
Tools were initially lacking to study the function of Thou proteins in the modulation of calcium channels and neurotransmitter release, just this changed for me when I visited Pietro De Camilli in Milan and attended the Fifth International Conference on Circadian Nucleotides and Protein Phosphorylation, in July 1983. There I heard almost the use of pertussis toxin to distinguish between different GTP binding poly peptide mechanisms. Although information technology was not still available commercially, I managed to locate a local source of pertussis toxin (entailing rather scary visits to the UK government Center for Applied Microbiology & Research at Porton Downwardly, where research on dangerous toxins and pathogens was conducted). Using both pertussis toxin and nonhydrolyzable guanine nucleotide analogs (including photoactivatable forms, made past John Wootton and David Trentham at NIMR), nosotros then investigated the involvement of pertussis toxin-sensitive G proteins in the modulation of both glutamate release (Dolphin and Prestwich, 1985) and calcium currents (Dolphin et al., 1986; Scott and Dolphin, 1986; Dolphin et al., 1988).
1 of the next big scientific questions was to examine the same modulatory processes using the calcium channel subunits that were being cloned at that time by several groups (Tanabe et al., 1987; Ellis et al., 1988; De Jongh et al., 1990; Mori et al., 1991; Starr et al., 1991; Williams et al., 1992). In 1989, I was asked to utilise for the Chair of Pharmacology at the Regal Gratuitous Infirmary School of Medicine in London, and in that location I reoriented our efforts to examine the function of specific calcium channel subunits in the modulation of native and cloned calcium currents past G proteins. In our piece of work, we concentrated initially on the part of the calcium aqueduct β subunits (Berrow et al., 1995). We then uncovered an essential role for the Due north-terminus of CaV2 calcium channels in their 1000-poly peptide modulation (Folio et al., 1998; Canti et al., 1999), and we demonstrated the importance of calcium channel β subunits in the Thousand-protein modulation of CaV2.2 channels (Meir et al., 2000; Leroy et al., 2005), and also in PI3 kinase-mediated calcium channel modulation (Viard et al., 2004).
At that fourth dimension, very trivial work had been done on the part of the intriguing α2δ subunits of calcium channels, whose topology and function were initially unclear (Brickley et al., 1995; Gurnett et al., 1996). We started working on these proteins through another fortunate encounter. When the Imperial Free School of Medicine was merged with Academy College London (UCL), I was asked to move to the UCL Gower Street campus in 1997, primarily to free up my infinite for Professor Geoff Burnstock, who had relinquished his office as Head of Department of Beefcake at UCL. There, I had the good fortune to collaborate with Michele Rees in the Pediatrics Section, on the molecular basis for the Ducky mouse mutation, that causes absence epilepsy and ataxia. The mutation turned out to be in a calcium channel auxiliary subunit factor, Cacna2d2, encoding αtwoδ−2 (Barclay et al., 2001). This piece of work fortuitously led the states on to an extended series of studies on the biochemistry, physiology, and pharmacology of the α2δ subunit family, about which lilliputian was then known. These investigations allowed me to combine my interests in biochemistry, from my undergraduate years, with all the many other techniques we take embraced, including electrophysiology. Amid our findings were that the von Willebrand cistron domain of α2δ subunits is essential for their ability to augment calcium currents (Canti et al., 2005; Hoppa et al., 2012; Dahimene et al., 2018), that α2δ proteins are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol linkage rather past a transmembrane domain (Davies et al., 2010), that the αtwoδ subunits increase trafficking of calcium channels to the cell surface (Cassidy et al., 2014), and that proteolytic cleavage of αiiδ into α2 and δ is an essential molecular switch for the augmentation of calcium currents and transmitter release (Kadurin et al., 2016; Ferron et al., 2018).
Other studies, including piece of work from our own laboratory, constitute that αtwoδ−1 mRNA and protein are strongly upregulated in DRG neurons in rodent models of neuropathic pain (Luo et al., 2001; Newton et al., 2001; Bauer et al., 2009). Furthermore, α2δ−1 is the main receptor for the antiepileptic drug gabapentin, which is also of therapeutic benefit in neuropathic hurting (Gee et al., 1996). We contributed to showing that their binding to αtwoδ−1 is essential for the activity of gabapentinoids to alleviate neuropathic pain (Field et al., 2006). Since α2δ proteins are auxiliary subunits, the molecular mechanism of action of these drugs in neuropathic pain was not immediately obvious, until we showed that gabapentinoids reduced calcium currents chronically, but not acutely, through an inhibitory consequence on α2δ trafficking, both in vitro (Hendrich et al., 2008; Tran-Van-Minh and Dolphin, 2010) and in vivo (Bauer et al., 2009). We then constitute, past making a functional extracellularly tagged version of CaV2.2, that as a consequence, gabapentin also reduced the trafficking and jail cell surface expression of the channel itself (Cassidy et al., 2014). A knock-in mouse containing this tagged Ca5two.2 is now allowing usa to place that αiiδ−1 KO also reduces the cell surface localization of CaVii.2 in DRGs and their presynaptic terminals in vivo (Nieto-Rostro et al., 2018).
The molecular mechanisms involved in the development and maintenance of neuropathic pain are the subject of research of many groups (for review, encounter Sexton et al., 2018; Alsaloum et al., 2020; Halievski et al., 2020). Equally a event of orienting our enquiry toward the involvement of calcium channels in neuropathic pain, I encountered another unexpected advantage of moving to UCL. We accept been able to benefit enormously from the knowledge and advice of the many experts in pain who work there, including John Wood, Tony Dickenson, and Maria Fitzgerald. Information technology is remarkable that, during my time at UCL, threads from my past have been picked upward and helped to shape my work. Indeed, we have collaborated extensively with Tony Dickenson, who was one of the other nontenured staff scientists who left NIMR at the same fourth dimension every bit me, and who so obtained a lectureship directly at UCL (Bauer et al., 2009; Patel et al., 2013). Similarly, in an extension of the Greengard family, we had a valuable collaboration with Tim Ryan, who has also worked extensively with Paul Greengard'southward laboratory, especially on the role of Synapsins in vesicular release (Ryan et al., 1996; Chi et al., 2003). With Tim, we studied the office of αtwoδ proteins in presynaptic Catwo+ entry and vesicular release (Hoppa et al., 2012), and this led us on to farther examining the role of proteolytic cleavage of αtwoδ in these processes (Kadurin et al., 2016; Ferron et al., 2018).
I was honored to speak at Paul's 80th birthday celebration, and then besides to attend his 90th birthday symposium, both held at Rockefeller University, where I was very happy to come across up with so many previous colleagues (Fig. 2). At the latter coming together, I also had the privilege of being able to tell Paul of my election to the Royal Order, and to give thanks him for his support; and I could see his real pleasure in this news. Although I accept never worked directly on Synapsins since leaving Paul'south laboratory, from these and other meetings, I have of form retained a keen interest in Paul'southward broad-ranging research, maintained until his death at age 93 in 2019. His work unraveling the enormous complexity of the many pathways involving neuronal protein phosphorylation, and their physiological and pathologic roles (Hilfiker et al., 1999; Svenningsson et al., 2004; Brichta and Greengard, 2014), contributed to his existence awarded the Nobel Prize in 2000, together with Arvid Carlsson and Eric Kandel.
For younger scientists, I have but a few messages. Like me, I hope y'all will discover that all your scientific studies and feel will be of benefit in the long run, fifty-fifty if you change fields, and all your scientific mentors, colleagues, and friends volition remain important throughout your career; but the value of take a chance, casual conversations, and fortuitous meetings cannot be overstated. Treasure them all.
Footnotes
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The author declares no competing financial interests.
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This work was supported past multiple grants from the United Kingdom Medical Research Council and the Wellcome Trust during my career. I give thanks Eric Nestler for reading an before version of this manuscript.
- Correspondence should exist addressed to Annette C. Dolphin at a.dolphin{at}ucl.ac.great britain
Source: https://www.jneurosci.org/content/41/10/2070
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