The Memory Board at Cold Spring Harbor Laboratory - Seven Memories of Cold Spring Harbor

SEVEN MEMORIES FROM COLD SPRING HARBOR

Jean-Pierre Changeux

Institut Pasteur & Collège de France Paris

The recollections I have of Cold Spring Harbor Laboratory are, if I may say, the most moving ones of my many visits to the United States. Cold Spring Harbor was my first contact with America when, in 1961, I attended the meeting on Cellular Regulatory Mechanisms . I was amazed to discover the New World as a charming miniature harbor with colonial houses scattered in a wood landscape. Nothing to do with the sky scrapers and the overdeveloping industries and factories. I was immediately embedded in the spirit of the founding fathers, Benjamin Franklin, Thomas Jefferson and my favorite philosophers of french enlightement. Also, it was my first speech as a young graduate student at an international meeting on a subject which was to become a major topic of protein chemistry and regulation : the concept of allosteric site and allosteric interaction. This was the start of an unchartered scientific trajectory in molecular pharmacology and neuroscience actually beaconed by the six successive presentations I gave at Cold Spring Harbor Symposia from 1961 to 1996, until my last visit in May 2006, 45 years later, in the friendly atmosphere of an informal debate on esthetic perception and consciousness under the instigation of Suzanne Nalbantian and Jan Witkowski.

The 1961 Symposium was somewhat an historical one in the field of molecular biology. It was devoted mainly to protein synthesis and its regulation. Its highlights were the Jacob and Monod model of the operon and the Brenner and Gros presentations of the first hints on messenger RNA. My presentation followed Umbarger talk on the feed back regulation of the activity of the bacterial enzyme : l-threonine deaminase, an interesting feature he had recently discovered. It was dedicated to a basic question of chemistry or theoretical enzymology raised by his findings : the actual elementary mechanism of the interaction between substrate and feedback regulatory signal taking place at the level of l-threonine deaminase. Following the presentation of my experimental results which demonstrated the uncoupling between catalytic activity and feedback regulation, I proposed two models : the standard overlapping sites with steric competition between the two types of ligands and, an alternative, new model of non-overlapping sites which postulated two distinct sites : one for the enzyme substrate and the other for the feed-back regulatory ligand where the interaction was assumed to be at distance or indirect. The data I showed supported the second scheme. My talk was praised and reformulated in more general terms in the Concluding remarks of the Symposium given orally by Jacques Monod at the end of the meeting. In the subsequently written chapter by Monod and Jacob the non-overlapping sites model was baptized allosteric , a word which was not pronounced during the meeting but emerged from lively debates back at the Pasteur Institute during the writing of the text. Worthnoting anecdote, at the meeting, immediately following my oral presentation, Bernard Davis stood up and suggested an analogy between l-threonine deaminase cooperative behaviour and hemoglobin cooperative binding of O2. This was a profound anticipation of the subsequent evolution of our thinking and of what was going to be the two-state concerted model of Monod Wyman and Changeux (1965) (MWC model) (rev. Changeux and Edelstein, 2005a).

Two years later, in 1963, the fast growing field of molecular biology justified a novel meeting on a closely related topic : the Synthesis and structure of macromolecules centered again on DNA, RNA and protein synthesis. Berg, Baltimore, Littauer talked about RNA, Rich and Gros about protein synthesis, Jacob and Brenner proposed the replicon model, Epstein described conditioned lethal mutations. Nirenberg and Ochoa revealed the secrets of the genetic code. Allosteric proteins now occupied a whole section of the meeting with as speakers Koshland, Gerhard and Pardee, Wyman, Tomkins. I gave a talk on my last findings on the allosteric interactions mediated by L-threonine deaminase. The new data confirmed my 1961 early observations for indirect interaction between sites but brought novel information : first the dicovery of allosteric activators of l-threonine deaminase and the linkage of positive cooperative interactions with heterotropic interactions with structurally different ligands : allosteric inhibitors and activators. Furthermore in the recently written review paper by Monod, Changeux & Jacob (1963) now entitled Allosteric proteins and cellular control systems, the possibility was mentioned that the conformational transition coupling the various sites on an allosteric protein was a transition of agregation. The idea originated from the data of Edmond Fischer on phosphorylase and from experiments that Jacques Monod was doing in the lab with Agnès Ullman on the same system. In fact, I had never observed anything like that with L-threonine desaminase. Yet, I found that if the enzyme was mildly inactivated by urea, a reversible equilibrium between an active and an inactive form of the enzyme became established. It was interpreted as an equilibrium between an active polymer and an inactive monomer . Moreover, allosteric effectors were found to displace this equilibrium in a rather interesting fashion : substrate and positive effectors enhanced inactivation, allosteric inhibitors, on the other hand, had the opposite strengthening effect. In the presentation I gave at the meeting I concluded that the allosteric transitions involve weakening or increasing interactions between subunits , that negative interactions between subunits pre-exist in the uncombined molecules which are differentially relaxed and stabilized by positive or negative effectors. This was to become one of the basic ingredients of the two-state mechanism subsequently postulated by Monod Wyman and Changeux in their 1965 theoretical paper On the nature of allosteric transitions to account for signal transduction in allosteric proteins.

Meanwhile, following a very fruitful and creative stay as a postdoctoral fellow in David Nachmansohn laboratory at Columbia University College of Physicians & Surgeons (from February to August, 1967), I decided to shift my scientific orientation to pharmacology and neurobiology. In my thesis work (1965), I had made the then far-fetched hypothesis that synaptic transmission involves allosteric mechanisms at the membrane level . But to make sense of this possibility, the still enigmatic receptor for the neurotransmitter involved in synaptic transmission had to be identified. Thetopic of the1975 Cold Spring Harbor Symposium was «the Synapse . The first meeting of the Association for Neuroscience had taken place a few years before (1971). The Neuroscience was on the rise. At the meeting, Paul Greengard demonstrated the role of phosphorylations in the brain, Hubel and Wiesel, Zéki discussed the visual system, Poggio, Marr and Stent closed the symposium with Model systems . My presence at the meeting was likely to be due to the identification of the acetylcholine receptor using snake venom toxins (Changeux, Kasai & Lee, 1970). But my own justification was also to examine a possible homology between the newly discovered acetylcholine receptor and the now classical allosteric proteins. I reviewed our first structural observations by electron microscopy of the receptor as an integral membrane protein. I also dicussed data on the multiple states of affinity of the receptor protein proposing to relate these affinity states with the states of opening of the ion channel. The conclusion I drew was that the low affinity states are activatable while the high affinity ones are desensitized with the ion channel shut. Furthermore, the conformational transitions of the membrane bound receptor were demonstrated with the fluorescent probe quinacrine. Interestingly, the two-state MWC model fitted the data as long as an additional third state was included. Unambiguously, the nicotinic receptor was a membrane bound regulatory protein yet with more complex features than the standard globular allosteric proteins. To the two-state mechanism for channel opening was to be added a slow transition of desensitization initially discovered in vivo by Langley (1905) and recorded as an electrophysiological trace by Katz and Thesleff in 1957. The reaction was now demonstrated in chemical and macromolecular terms as a conformational transition towards a very high affinity closed state.

The 1983 Symposium on Molecular Neurobiology was a rather tense and competitive one, yet quite stimulating. It was placed under the auspices of molecular cloning and sequencing. Edelman presented his data on the first cell adhesion molecule ; Brenner, Sulston, Horowitz illustrated the multiple advantages of the worm Caenorhabditis for the neuroscientist. J. Mallet had cloned and sequenced Tyrosine hydroxylase, the first enzyme of catecholamine biosynthesis. The introductory section of the meeting was on the nicotinic receptor. All the main protagonists of the field were present : Karlin, Raftery, Patrick, Heinemann, Barnard and Numa. The competition had been fierce in the past months for the first complete sequence of the nicotinic receptor. We had brought the first amino acid sequence data for the nicotinic receptor with Torpedo marmorata a-subunit, which was quickly confirmed and extended by Raftery group. Yet, we were not fast enough to get the complete sequence first. We got it a few months after Numa with T. marmora a-subunit. On the other hand, faithfull to my philosophy, the data I presented were focussed more on the understanding of structure-function relationship of the receptor protein than on the collection of brute force sequence data. The results I presented dealt of course with the allosteric properties of the receptor. Retrospectivelly, they were to become crucial, a few years later, in the identification of the ion channel and the demonstration of its allosteric interactions with the acetylcholine binding site. Indeed, a new site for non-competitive blockers was characterized by radioactive/fluorescent ligand binding and shown to behave as an allosteric site interacting in a positive and reciprocal manner with the acetylcholine binding site. The receptor not only behaved as an authentic membrane allosteric protein but the identification of the biologically active site , the ion channel was on the way. Indeed, one of these noncompetitive blockers, chlorpromazine, was shown, a few years later in my laboratory, to label an amino acid (d-Ser 262) from the membrane segment MII of the nicotinic receptor d-subunit (Giraudat et al 1986), thus pointing to this membrane domain as a component of the ion channel. This last finding was rapidly confirmed and extended in vivo by Numa, Sakmann and their colleagues using the patch-clamp method and the frog oocyte as an expression system. The allosteric transitions of the nicotinic receptor could now be investigated at the amino acid level (reviewed in Changeux 1990; Changeux & Edelstein 2005b).

The 1990 Cold Spring Harbor meeting on the Brain signed the success of the Neuroscience. The signal transduction mechanisms invaded the field, the molecular biology of synapse development became accessible. Cognitive sciences entered neuroscience with the bugle call of Crick and Koch on their Neural correlates of consciousness . Damasio and Rolls but also Poggio and Shimon Ullmann were there. I was in section 2 and presented our molecular biology data on the compartimentalized expression of the nicotinic receptor during the development of the neuromuscular junction. This was the first presentation of the promoter sequence of a receptor subunit gene and the identification of the first DNA elements and transcription factors (MyoD sites) involved in this compartmentalisation. Moreover, we showed that following the transcriptional regulation, a posttranscriptional mechanism mobilized a secretory pathway targeting the nascent receptor protein to the postsynaptic membrane with specialized Golgi apparatus and microtubules and a clustering stabilisation of the receptor protein thanks to the 43K protein initially discovered in our laboratory (but subsequently rebaptided RAPSYN by Merlie and Cohen). The supra-molecular organisation of the synaptic membrane was on the way.

My last invitation to a Cold Spring Harbor Symposium was in 1996 on the Function and dysfunction in the nervous system . My presentation was entitled Nicotinic receptor and brain plasticity . The emphasis had shifted from the electric organ and neuromuscular junction receptors to brain nicotinic receptors. The target molecule was still (and always) the nicotinic receptor. The philosophy had not changed. It was still to relate structure and function. But the functions were now the elusive and often controversial higher functions of the brain. To reach this new universe, one had to climb carefully and sometimes painfully through the successive levels of organisation bridging the molecular and the cognitive levels. The starting blocks were the diverse nicotinic receptor subunit genes and oligomers, but also, as expected, the particular features of the allosteric transitions of brain receptors. First, I suggested that brain nicotinic receptors may contribute to Ca++ allosteric switches : they permeate Ca++ ions to an extent such that some of them (a7) rivalizes on this feature with NMDA receptor, also, they are activated by external Ca++ ions when they bind to specific allosteric sites. Second, the regional and neural network distribution of nicotinic receptor subunit genes are governed by specific promoter elements and transcription factors, yet, the combinations involved are far more complex than those which account for the development of the neuromuscular junction. Third, a subcellular compartimentalisation takes place as well in the distribution of brain nicotinic receptors, but, here again, in more diversified and complex patterns : in the somato-dendritic compartment, in the axonal terminal, but also, as postulated decades before by Nachmansohn, along the axon, in a pre-terminal compartment. Fourth, nicotinic receptors in prefrontal cortex are electrophysiologically functional and their pharmacological blocking interferes with the performance of cognitive tasks in the rat. Moreover the deletion of high affinity b2-containing subunit in the mouse alters avoidance learning as well as nicotine reward and nicotine mediation of dopamine release. Computational models of neuronal architectures able to pass cognitive tasks (like delayed-response tasks, Wisconsin card sorting tasks) though selection by reward had been elaborated together with Stanislas Dehaene. They included nicotinic receptors as coincidence detectors. The nicotinic receptor was ready to become a useful tool to bridge the molecular and the cognitive levels in the brain (rev. Changeux 2006).

My last visit to Cold Spring Harbor, in May 2006, was not related to a Symposium. I gave two talks, one talk was a formal seminar in the same room now called the operon where I gave my first presentation in 1961. It aimed at a concrete, down to earth, demonstration that, indeed, as postulated almost 40 years ago, the nicotinic receptor is an authentic allosteric membrane protein. But also that if its allosteric properties account for signal transduction and fast plasticity of brain synapses, they also explain some forms of neuropathological diseases, like congenital myasthenia or autosomal dominant frontal lobe epilepsia. The gain of function mutations in nicotinic receptor genes that unambiguously demonstrate the allosteric properties of the receptor protein, may serve as adequate models of brain dysfunctions. And this conclusion may be legitimately generalized to other categories of receptors like the G-protein linked receptors, thus introducing the concept of allosteric disease . Also, they pave the way to a new exciting and still far fetched issue: the molecular biology of consciousness!

The second talk was an informal morning discussion introduced by Suzanne Nalbantian, a pioneer in the attempt to fill the gap between neuroscience and humanities (a courageous move indeed in an outstanding environment of molecular and cellular neuroscience). The aim was to foster the idea of a neuroscience of esthetics … I accepted the challenge wholeheartedly. Yet at the risk to tarnish my scientific reputation, I suggested that, in addition to the much studied mechanisms of sensory (in particular visual) perception, one should now consider in the case of esthetic perception the issue of access to consciousness. This program becomes a plausible one in the framework of, for instance, the models proposed by Dehaene and myself referred to as the neuronal workspace hypothesis . The idea of a coherent access of sensory, emotional and long term memory traces to the conscious workspace was debated as one (among many other) possible neural mechanism of esthetic experience. Contrary to my expectations, the debate was warm, friendly and constructive. The scientific challenge appeared to be real!

In retrospect, these seven visits to Cold Spring Harbor laboratory had an immense stimulating effect on the evolution of my thinking. But, the impact of the Cold Spring Harbor Symposium is certainly much broader than my own feelings ! Many scientists, I am sure, share the same view. The Cold Spring Harbor Symposia played, and still play, a major contribution to the progress of science at its cutting edge. I wish that after 40 years the time has not yet come for me to say Good bye Cold Spring Harbor but only au revoir …