Monday, October 20, 2014

Interview with Robert D. Stolorow (2011, at Figure/Ground)

If I could study with any living therapist, Robert Stolorow is one of three people I would pay to learn from (the others are Donna Orange [a frequent Stolorow collaborator] and Diana Fosha). [Well, okay, there is a fourth one, another frequent Stolorow collaborator, George Atwood.] Throughout his career, he has emphasized the phenomenological experience of the client in psychotherapy, often invoking philosophers such as Heidegger or Gadamer, and the intersubjective space created in the therapeutic dyad. 

I have read nearly all of his books, as well as a LOT of his articles. There is a near-full listing of his books in the introduction to the interview - of those (any one is a great read), I highly recommend World, Affectivity, Trauma: Heidegger and Post-Cartesian Psychoanalysis (2011), Working Intersubjectively: Contextualism in Psychoanalytic Practice (1997), Structures of Subjectivity: Explorations in Psychoanalytic Phenomenology (2014 [1984]. 2nd ed.), and The Intersubjective Perspective (1994), the latter being a collection of essays edited by Stolorow, George Atwood, and George Brandchaft.

Here is a passage from World, Affectivity, Trauma: Heidegger and Post-Cartesian Psychoanalysis (p. 44):
Trauma shatters the absolutisms of everyday life, which, like the illusions of the “they,” evade and cover up the finitude, contingency, and embeddedness of our existence and the indefiniteness of its certain extinction. Such shattering exposes what had been heretofore concealed, thereby plunging the traumatized person, in Heidegger’s terms, into a form of authentic Being-toward-death and into the anxiety—the loss of significance, the uncanniness—through which authentic Being-toward-death is disclosed. Trauma, like authentic Being-toward-death, individualizes us, but in a manner that manifests in an excruciating sense of singularity and solitude.
And this (p. 55-56):
Trauma devastatingly disrupts the ordinary, average-everyday linearity and “ecstatical unity of temporality” (Heidegger, 1927, p. 416), the sense of “stretching-along” (p. 426) from the past to an open future. Experiences of emotional trauma become freezeframed into an eternal present in which one remains forever trapped, or to which one is condemned to be perpetually returned through the portkeys supplied by life’s slings and arrows. In the region of trauma, all duration or stretching along collapses; past becomes present, and future loses all meaning other than endless repetition. In this sense it is trauma, not, as Freud (1915) would have it, the unconscious that is timeless.
Because trauma so profoundly modifies the universal or shared structure of temporality, the traumatized person quite literally lives in another kind of reality, an experiential world felt to be incommensurable with those of others. This felt incommensurability, in turn, contributes to the sense of alienation and estrangement from other human beings that typically haunts the traumatized person. Torn from the communal fabric of being-in-time, trauma remains insulated from human dialogue.
In the first paragraph, "portkeys" is a Harry Potter reference (that I was unaware of) and are defined as "objects that transported him instantly to other places, obliterating the duration ordinarily required for travel from one location to another."

Anyway, I could go on all day quoting Stolorow's texts and my appreciation for them.

All of this serves as an introduction to an interview of Stolorow from Figure/Ground back in 2011.

Interview with Robert D. Stolorow

© Robert D. Stolorow and Figure/Ground
Dr. Stolorow was interviewed by Laureano Ralón. June 13th, 2011.

Robert D. Stolorow, Ph.D., is a Founding Faculty Member and Training and Supervising Analyst at the Institute of Contemporary Psychoanalysis, Los Angeles; a Founding Faculty Member at the Institute for the Psychoanalytic Study of Subjectivity, New York City; and a Clinical Professor of Psychiatry at the UCLA School of Medicine. He is the author of World, Affectivity, Trauma: Heidegger and Post-Cartesian Psychoanalysis (2011) and Trauma and Human Existence: Autobiographical, Psychoanalytic, and Philosophical Reflections (2007), and co-author of Worlds of Experience: Interweaving Philosophical and Clinical Dimensions in Psychoanalysis (2002), Working Intersubjectively: Contextualism in Psychoanalytic Practice (1997), Contexts of Being: The Intersubjective Foundations of Psychological Life (1992), Psychoanalytic Treatment: An Intersubjective Approach (1987), Structures of Subjectivity: Explorations in Psychoanalytic Phenomenology (2014 [1984]. 2nd ed.), Psychoanalysis of Developmental Arrests: Theory and Treatment (1980), and Faces in a Cloud: Intersubjectivity in Personality Theory (1993 [1979], 2nd. ed.). He is also coeditor of The Intersubjective Perspective (1994) and has authored or coauthored more than two hundred articles on aspects of psychoanalytic theory and practice. He received his Ph.D. in Clinical Psychology from Harvard University in 1970 and his Certificate in Psychoanalysis and Psychotherapy from the Psychoanalytic Institute of the Postgraduate Center for Mental Health, New York City, in 1974. He also received a Ph.D. in Philosophy from the University of California at Riverside in 2007. He holds diplomas both in Clinical Psychology and in Psychoanalysis from the American Board of Professional Psychology (ABPP). In 1995 he received the Distinguished Scientific Award from the Division of Psychoanalysis of the American Psychological Association, in which he is a Fellow.

What would you define yourself as – an author, a thinker, a public intellectual?

Being a weird interdisciplinary creature, I have to define myself somewhat complexly. I definitely think of myself as a psychoanalytic and philosophical thinker and author. Additionally, I am a practitioner of psychoanalysis and a teacher of both philosophy and psychoanalysis. In recent times, I have also been publishing articles and blogs applying my ideas about collective trauma and defensive ideologies to the socio-political sphere, so I guess that might make me a public intellectual too.

Who were some of your mentors in university and what were some of the most important lessons you learned from them?

I earned a doctorate in clinical psychology at Harvard in 1970 and a doctorate in philosophy at the University of California, Riverside, in 2007, and I reaped rich benefits from mentors during both periods of graduate study. My principal mentor at Harvard was Robert White, from whom I acquired an abiding interest in and respect for the uniqueness of each individual’s world of experience. My principal mentor at Riverside was my dissertation chair, William Bracken, who, although relatively unpublished, is perhaps the most brilliant Heidegger scholar I have encountered. I owe him an enormous dept of gratitude for his contributions to my development as a Heideggerian philosopher. Other important mentors at Riverside from whom I learned a great deal were Kantian philosopher Andrews Reath, phenomenologist Charles Siewert, and another brilliant Heidegger scholar, Mark Wrathall.

You trained both as a philosopher and a psychoanalyst. How did the two careers reinforce each other?

Wow, I would have to write an intellectual memoir to address this question adequately! I’ll try to hit the highlights. I first became interested in the interface of psychoanalysis and philosophy as an undergraduate in the early 1960s when I encountered the writings of Ludwig Binswanger, Medard Boss, and Rollo May, early pioneers who recognized the relevance of Heidegger’s existential philosophy for psychotherapy and psychoanalysis. While a graduate student in clinical psychology, I became disillusioned with empirical psychological research, feeling that it stripped psychology of everything humanly meaningful, and toyed with the idea of doing a second doctorate in philosophy (an ambition that had to await several decades before coming to fruition), which at the time I thought could provide tools for cleaning up the mess that was psychoanalytic theory. However, during my clinical internship I found that I really enjoyed psychoanalytic work and, after completing my doctorate, decided to go to New York to pursue psychoanalytic training instead.

A nodal point in my intellectual career occurred in 1972 when, still in psychoanalytic training, I took a job as an assistant professor of psychology at Rutgers where I met George Atwood, who became my closest collaborator. George (an autodidact with an encyclopaedic knowledge of Continental philosophy) and I embarked upon a series of psycho-biographical studies of the personal, subjective origins of the theoretical systems of Freud, Jung, Rank, and Reich, studies that formed the basis of our first book, Faces in a Cloud: Subjectivity in Personality Theory (Aronson, 1979). From these studies, we concluded that since psychological theories derive to a significant degree from the subjective concerns of their creators, what psychoanalysis and personality psychology needed was a theory of subjectivity itself: a unifying framework capable of accounting not only for the psychological phenomena that other theories address, but also for the theories themselves. In the last chapter of Faces, we outlined a set of proposals for the creation of such a framework, which we called psychoanalytic phenomenology. We envisioned this framework as a depth psychology of personal experience, purified of the mechanistic reifications of Freudian meta-psychology. Our framework took the experiential world of the individual as its central theoretical construct. We assumed no impersonal psychical agencies or motivational prime movers in order to explain the experiential world. Instead, we assumed that this world evolves organically from the person’s encounter with the critical formative experiences that constitute his or her unique life history. Once established, it becomes discernible in the distinctive, recurrent patterns, themes, and invariant meanings that pre-reflectively organize the person’s experiences. Psychoanalytic phenomenology entailed a set of interpretative principles for investigating the nature, origins, purposes, and transformations of the configurations of self and other pervading a person’s experiential world. Importantly, our dedication to illuminating personal phenomenology had led us from Cartesian minds to emotional worlds and, thus, from intra-psychic mental contents to relational contexts. Phenomenology had led us inexorably to contextualism.

Once we had rethought psychoanalysis as a form of phenomenological inquiry, a focus on the mutually-enriching interface of psychoanalysis and Continental phenomenology became inescapable, and I began reading phenomenological philosophy voraciously. In 2000, I formed a leaderless philosophical study group in which we devoted a year to a close reading of Heidegger’s Being and Time and another year to Gadamer’s Truth and Method. Philosopher-psychoanalyst Donna Orange had joined the collaboration with Atwood, and she brought to our phenomenological contextualism a perspectivalist hermeneutic sensibility and a view of psychoanalytic practice as a form of phronesis rather than techne.

A second nodal point for me occurred when I turned my attention to the phenomenology of emotional trauma in the wake of the death of my late wife, Dede, in 1991—a massive trauma that shattered my world. The close study of Being and Time in 2000 proved to be critical. On one hand, Heidegger’s ontological contextualism (In-der-Welt-sein) seemed to provide a solid philosophical grounding for our psychoanalytic phenomenological contextualism. Even more important to me at the time, Heidegger’s phenomenological analysis of Angst, world-collapse, uncanniness, and thrownness into being-toward-death provided me with extraordinary philosophical tools for grasping the existential significance of emotional trauma. It was this latter discovery that motivated me to begin doctoral studies in philosophy and write a dissertation on trauma and Heidegger, which eventuated in my two most recent books, Trauma and Human Existence: Autobiographical, Psychoanalytic, and Philosophical Reflections (Routledge, 2007) and World, Affectivity, Trauma: Heidegger and Post-Cartesian Psychoanalysis (Routledge, 2011). In the last book, I showed both how Heidegger’s existential philosophy can ground and enrich post-Cartesian psychoanalysis and how post-Cartesian psychoanalysis, by relationalizing Heidegger’s conception of finitude and expanding Heidegger’s conception of relationality, can enrich his existential philosophy. I feel that in this book I have, in my sunset years, come into my own as a philosopher.

In your experience, how do you think the role of university professor might have evolved since you were an undergraduate student?

Perhaps partly because I have not been a university professor (in psychology) since 1984 when I moved to California, I have not noticed significant changes in the role of university professor. I was very struck by the enormous devotion to teaching, guiding, and mentoring shown by my philosophy professors at Riverside. Perhaps the biggest change for me as a graduate student was the current importance of the internet and the need for me to become computer-literate fast!

How do you manage to command attention during your talks and lectures in this “age of interruption” characterized by fractured attention and information overload?

When I first began lecturing and then presenting in the early 1970s, I learned to bring my affect into my speaking. This has served me well ever since. I have found that the affect-laden quality of my recent work has been especially appealing to young philosophers.

The following guest question was drafted by Professor Iain Thomson: “Do you think all resurrective ideologies necessarily deny human finitude? What about the later Heidegger’s postmodern idea that truly acknowledging human finitude can give us insight into the inexhaustible nature of being?”

This is a great question. There have been two contexts in which I have written about “resurrective ideology.” One has been my effort to extend my ideas about trauma to the socio-political sphere. In my 2007 book on trauma, I contended that the essence of emotional trauma lies in the shattering of what I called the “absolutisms of everyday life,” the system of illusory beliefs that allow us to function in the world, experienced as stable, predictable, and safe. Such shattering is a massive loss of innocence exposing the inescapable contingency of existence on a universe that is chaotic and unpredictable and in which no safety or continuity of being can be assured. Emotional trauma brings us face to face with our finitude and existential vulnerability and with death and loss as possibilities that define our existence and that loom as constant threats. Often traumatized people try to restore the lost illusions shattered by trauma through some form of resurrective ideology.

Consider, for example, the impact on Americans of the terrorist attack of September 11, 2001, a devastating collective trauma that inflicted a rip in the fabric of the American psyche. In horrifyingly demonstrating that even America can be assaulted on its native soil, the attack of 9/11 shattered Americans’ collective illusions of safety, inviolability, and grandiose invincibility, illusions that had long been mainstays of the American historical identity. In the wake of such shattering, Americans became much more susceptible to resurrective ideologies—e.g., that offered by the Bush administration—that promised to restore the grandiose illusions that have been lost.

The other context, actually the original one, was a psycho-biographical account of Heidegger’s fall into Nazism, which I wrote in collaboration with Atwood and Orange and incorporated into my 2011 book. There we contended that Heidegger’s enthusiastic embrace of his version of Nazism, whose grandiose quality was chillingly manifested in his Rector’s Address, “The Self-Assertion of the German University” (1933), represented his effort to resurrect his sense of agentic selfhood, which had been crushed by the combined emotionally annihilating impact of three circumstances: His muse and lover Hannah Arendt’s withdrawal from him; his magnum opus Being and Time’s being met by the academic world “by hopeless incomprehension”; and his mother’s essentially disowning him on her deathbed for his having broken with the Catholic Church.

After resigning as rector of Freiburg University in 1934 and disengaging from political involvement, Heidegger largely withdrew into a life of solitary philosophical and spiritual reflection, wherein the “turn” in his thinking gained momentum. I think Iain Thomson is right when he claims that the later Heidegger’s acknowledgment and acceptance of an aspect of human finitude—namely, the historically and temporally embedded limitedness of any understanding of being—gave him insight into “being as such,” the inexhaustible source of all intelligibility that resists any attempt to conceptualize it. And yet, do we not glimpse a trace of the old restorative grandiosity in Heidegger’s self-designation as the agent of a new “other beginning,” the initiator of a new epoch in the history of being?

Other emotional themes in Heidegger’s later philosophy are apparent to a psychoanalytic eye. Heidegger is often rightly criticized for never having openly expressed remorse about his Nazi involvement. Yet the whole tenor of his later philosophizing—wherein the grandiose, aggressive, goose-stepping self-assertiveness of the Rector’s Address is replaced by a view of the human being as the “constant receiver,” the “shepherd” and the protector, of the “gift” of being—can be seen to reflect his recognition of his dreadful, deplorable mistake.

Moreover, there is another dimension of human finitude—the finitude of human connectedness, of our “being-with-one-another”—that goes largely unnamed throughout Heidegger’s philosophizing. In my 2011 book, I claimed controversially (with Critchley and Derrida) that human finitude is relational, that being-toward-death always includes a being-toward-loss of loved others, and that death and loss are existentially equiprimordial. In the chapter on Heidegger’s Nazism, we contended that for Heidegger the threat of loss of connectedness with others was built into the quest for authentic individualized selfhood, as was shown vividly in his wrenching struggles to separate himself from the Catholic Church of his family and in his mother’s deathbed renunciation of him for doing just that. In the poetry of Holderlin, Heidegger found the powerful theme of returning—returning to being-at-home and to the lost god that had disappeared—imagery in which we discerned his longing to restore connections lost in his pursuit of individualized selfhood, such as those with his mother and the Catholic family of his childhood. The later Heidegger returned home.

Returning for a moment to your dual training as a philosopher/psychoanalyst, do you think any insights from the social sciences might help transform the philosophical profession for the better and vice versa? Should fields like philosophy and psychology/sociology remain separate, or are there advantages to bridging the existential and existentielle dimensions of human reality in the spirit of interdisciplinary studies and methodological pragmatism?

Clearly, as an interdisciplinary creature myself, I am an advocate of interdisciplinary cross-fertilization (of which my 2011 book is a clear instance), rather than disciplinary insularity. Heidegger’s Being and Time is filled with examples of the advantages of bridging the existential and the existentielle, the ontological and the ontical dimensions of human reality. It is my view that academic psychology made a big historical mistake when, caught in the grip of modern scientism, it separated itself from philosophy in order to become a “hard science.” I regard psychoanalysis, or at least my brand of it, as being neither a branch of medicine nor of psychology, but as applied philosophy.

You have defined your intersubjective-systems theory as a “phenomenological contextualism.” How is your own brand of contextualism similar and/or different from the relational model put forth by social constructionist thought?

There are of course many similarities, but I think there are subtle differences—differences in sensibility—as well. I would say that my brand of contextualism embraces a hermeneutic rather than a constructivist sensibility. Following Gadamer, I would say that all understanding involves interpretation, and this seems different to me from saying that all understanding is constructed. Interpreting something—i.e., understanding it from a particular perspective—seems different to me from constructing a narrative about it.

I assume you are familiar with Speculative Realism and Object-Oriented Ontology. Since your approach to psychiatry is both phenomenological and contextual, I will quote a passage from Graham Harman’s Guerrilla Metaphysics and ask you to reflect on it: “What I am advocating is a reversal of the familiar social pattern in which everyone proves their adequate philosophical training by jabbing a few more daggers into the corpse of realism. From the flintiest analytic philosopher to the most dashing Francophone icon, philosophy today is united through a shared contempt for any probing of a real world in itself. Like all broad fashions of any era, this disdain begins to take on the character of an automatic reflex, and like all mental reflexes soon decays into compulsion. Given this atmosphere, it is widely supposed that substances are championed only by reactionaries living in an irrelevant past, while innovation seems to be on the side of relations and contexts, not individual things. On a related front, it is supposed to be the reactionaries who believe in substances independent of our perceptions, while the self-proclaimed avant-garde delights in bursting this final bubble of the true believers – a tedious drama of canned iconoclasm playing out across the decades. The champions of wholes over parts and the doubters of independent realities can continue to mock the conservatism of their foes if they wish, but the fact is that they have now largely defeated those foes. Holism and antirealism, their days of novelty long past, have become the new philosophical dogmas of our time. The sole difference is that the old orthodoxies viewed their opponents as dangerous cutting-edge transgressors, while the new ones have so exhausted the field of critique and transgression that they are likely to view their challengers only as conservative throwbacks.” Is metaphysics a thing of the past in your view, or do you tend to agree more with Harman?

I don’t really know whether metaphysics is a thing of the past. Heidegger certainly thought that it was, or wished it to be so. What I would say is that metaphysical questions, like the debate between realism and anti-realism, fall outside the domain of phenomenological inquiry (except insofar as metaphysical systems can be historically contextualized and deconstructed, as Heidegger attempted to do). I think Husserl got it right when he characterized the intentional structure of consciousness phenomenologically as always as if directed toward an object, where the “as if” indicates that the metaphysical question about the reality of the intentional object is not to be asked by the phenomenological inquirer.

In agreement with Nietzsche, Heidegger, and Gadamer, my own phenomenological-contextualist viewpoint holds that all understandings of the “real world” are deeply perspectival. A passage from my 2011 book makes this claim very strongly: “Corresponding to its Cartesianism is traditional psychoanalysis’s objectivist epistemology. One isolated mind, the analyst, is claimed to make objective observations and interpretations of another isolated mind, the patient. A phenomenological contextualism … reunites the Cartesian isolated mind with its world…. Correspondingly, intersubjective-systems theory embraces a perspectivalist epistemology, insisting that analytic understanding is always from a perspective shaped by the organizing principles of the inquirer. Accordingly, there are no objective or neutral analysts, no immaculate perceptions (Nietzsche), no God’s-eye view (Putnam) of anyone or anything” (p.20).

What are you currently working on?

I’m planning a paper elaborating on Heidegger’s use of mood as a bridge between the ontical or psychological and the ontological, a bridge to the “truth of being.” In this paper, I want to counter two criticisms of Heidegger: (1) that he fails to distinguish sufficiently the phenomena of mood, emotion, and feeling, and (2) that he neglects the ontological significance of the body.

What Do Mirror Neurons Really Do? (Brain Science Podcast 112, w/ Gregory Hickok)

Gregory Hickok is the author of The Myth of Mirror Neurons: The Real Neuroscience of Communication and Cognition (2014), and he was the guest on the most recent episode of the Brain Science Podcast, hosted by Dr. Ginger Campbell.

This is from the Amazon ad copy for the book:
In The Myth of Mirror Neurons, neuroscientist Gregory Hickok reexamines the mirror neuron story and finds that it is built on a tenuous foundation—a pair of codependent assumptions about mirror neuron activity and human understanding. Drawing on a broad range of observations from work on animal behavior, modern neuroimaging, neurological disorders, and more, Hickok argues that the foundational assumptions fall flat in light of the facts. He then explores alternative explanations of mirror neuron function while illuminating crucial questions about human cognition and brain function: Why do humans imitate so prodigiously? How different are the left and right hemispheres of the brain? Why do we have two visual systems? Do we need to be able to talk to understand speech? What’s going wrong in autism? Can humans read minds?

The Myth of Mirror Neurons not only delivers an instructive tale about the course of scientific progress—from discovery to theory to revision—but also provides deep insights into the organization and function of the human brain and the nature of communication and cognition.
Sounds like a great book and an important counter-balance to all of the hype being piled on the concept of mirror neurons since their discovery.

What Do Mirror Neurons Really Do? (BSP 112)

October 16, 2014/ Ginger Campbell, MD

Greg Hickok, PhD 

Ever since their chance discovery back in 1992 mirror neurons have captured the imagination of both scientists and nonscientists, but their actual role remains mostly speculative. In The Myth of Mirror Neurons: The Real Neuroscience of Communication and Cognition, Dr. Gregory Hickok (UC-Irvine) explains why the most popular theory is probably wrong. He also provides a fascinating account of how science is really done and the sobering lesson that scientists can fall prey to the same cognitive biases (and tendencies toward laziness) that plague all humans.

I first discussed the discovery of mirror neurons back in BSP 35 when I featured Mirrors in the brain: How our minds share actions, emotions, and experience (2008) by Giacomo Rizzolatti and Corrado Sinigaglia. At that time what I found most fascinating was that since mirror neurons fire both when a subject (usually a monkey) performs an action and when a similar action is observed, this proves that single neurons are not necessarily purely motor or purely sensory. This surprising discovery seems to have been overshadowed in the rush to use mirror neurons to explain everything from autism to language evolution.

The latest Brain Science Podcast (BSP 112) features an interview with Dr. Gregory Hickok. BSP 35 is also available for FREE via the Brain Science Podcast Mobile APP.

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Sunday, October 19, 2014

Massimo Pigliucci: Scientia Salon - A Manifesto for 21st Century Intellectualism

Scientia Salon is a webzine about science and philosophy. There is a very active conversation occurring there around these two BIG topics. Back in March, Massimo Pigliucci (founder and editor of the site) posted a "manifesto for 21st Century intellectualism.

Here is a little taste . . .
That problem, to put it plainly, is that many of our fellow citizens have not taken any intellectual course in self-defense (literally or more broadly speaking), and that they are bombarded by the best political and — let’s not forget it — corporate propaganda money (a lot of money) can buy.

This has a cascade of effects, from the very personal to the societal. At the personal level, many go about their lives with hardly a clue about why they are doing what they are doing. Socrates’ dictum that an unexamined life is not worth living was surely an exaggeration, but it definitely pays from time to time to pause and reflect on what we want from our existence on this planet and why. The ancient called it the pursuit of eudaimonia, the good and moral life. But in order to reflect and make informed decisions we need thinking tools, and there is a dearth of them both inside and outside of our educational system.

At the societal level, this means that we elect politicians because they look like the sort of fellows one would want to have a beer with, rather than because they are honest and brimming with good ideas about how to navigate the perils of the modern world in ways that maximize fairness and well being.

The whole manifesto is posted below - be sure to check out some of the other writing at their site - some interesting topics and authors.

Massimo Pigliucci is, most recently, co-editor (with Maarten Boudry) of Philosophy of Pseudoscience: Reconsidering the Demarcation Problem (2013).

Scientia Salon: a manifesto for 21st century intellectualism

by Massimo Pigliucci
Arcimboldo's Librarian

During the Enlightenment, the Marquis de Condorcet defined a public intellectual as someone devoted to “the tracking down of prejudices in the hiding places where priests, the schools, the government, and all long-established institutions had gathered and protected them.” A number of years later, on 13 January 1898 to be precise, the writer Emile Zola showed the world — and in particular the French government — what public intellectualism could do. He penned his famous “J’accuse” letter to the President of France, concerning the abysmal behavior of the French authorities in the infamous Dreyfus affair.

Intellectualism, of course, has its detractors, particularly in the United States. Richard Hofstadter’s classic “Anti-Intellectualism in American Life” [1] traces several strands of the phenomenon all throughout American history, and we can very much see it today in the form of religious-based opposition to the teaching of evolution in public schools, or in the open disdain for politicians who dare name a favorite philosopher that is not Jesus.

At the City University of New York, where I work, I often teach a basic course in Critical Reasoning that uses a handy little booklet entitled, A Short Course in Intellectual Self Defense, by University of Québec-Montreal professor of Education Fundamentals Normand Baillargeon [2]. I highly recommend it, just as I wholeheartedly agree that teaching critical thinking really ought to be part of our concept of “educational fundamentals” — despite the fact that it is seen as optional even at the college level, let alone earlier.

The title of Baillargeon’s book is a play on a quote by the most famous (and controversial) public intellectual of the late 20th and early 21st century: Noam Chomsky. In his essay on Necessary Illusions: Thought Control in Democratic Societies [3], Chomsky wrote that “Citizens of the democratic societies should undertake a course of intellectual self defense to protect themselves from manipulation and control, and to lay the basis for meaningful democracy.” No kidding. Just turn on Fox News, MSNBC, or even CNN and you will see just how intense and damaging the political propaganda is in the US (I know it is no less so in the other country I am directly familiar with, Italy — and I doubt the situation is much different elsewhere, give or take).

People concerned with developing a thriving society have always been worried about the specter of totalitarianism, let’s call it the “1984” scenario. And to be sure, there is much of that still going on in the world, with billions of people living under non democratic (or democratic only in name) regimes, from China to Russia to much of the Middle East.

But a more subtle, arguably more effective, way of controlling people is rather akin to the scenario presented by Aldous Huxley in Brave New World. In that particular dystopia it is psychological (and biological) manipulation that plays the crucial role — and it is much more powerful than overt coercion. Or, if you prefer, arch back to the Roman empire, where its military might abroad was coupled with the famous panem et circenses approach to handling the civilian population at home: give them enough food and entertainment and they won’t have the stomach for a revolution. If that sounds as an even rough approximation of many modern societies, then we agree that we have a serious problem.

That problem, to put it plainly, is that many of our fellow citizens have not taken any intellectual course in self-defense (literally or more broadly speaking), and that they are bombarded by the best political and — let’s not forget it — corporate propaganda money (a lot of money) can buy.

This has a cascade of effects, from the very personal to the societal. At the personal level, many go about their lives with hardly a clue about why they are doing what they are doing. Socrates’ dictum that an unexamined life is not worth living was surely an exaggeration, but it definitely pays from time to time to pause and reflect on what we want from our existence on this planet and why. The ancient called it the pursuit of eudaimonia, the good and moral life. But in order to reflect and make informed decisions we need thinking tools, and there is a dearth of them both inside and outside of our educational system.

At the societal level, this means that we elect politicians because they look like the sort of fellows one would want to have a beer with, rather than because they are honest and brimming with good ideas about how to navigate the perils of the modern world in ways that maximize fairness and well being.

For all of this, I am not so naive as to propose that more education — or even more courses in critical thinking — is the panacea needed to cure the ills of humanity. After all, David Hume famously said that “Reason is, and ought only to be the slave of the passions, and can never pretend to any other office than to serve and obey them” [4], by which he meant that human beings aren’t just the rational animal (as Aristotle suggested), but also the passionate one. It follows that one needs to take care of emotions just as much, if not more, as one addresses reason.

Indeed, modern research in cognitive science has borne out Hume’s warning. Antonio Damasio [5] has argued in his Descartes’ Error that a functional human being cannot be modeled on the emotionless Spock, unless we wish for a polity of sociopaths. And of course more recent writings by the likes of Daniel Kahneman [6] and Jonathan Haidt [7] have clinched the case that we are so fraught with cognitive biases and so prone to rationalization that it is a miracle anything gets done around the world.

And yet, stuff does get done. Humanity has invented philosophy, and then science, and both have thrived precisely because we can and do use reason to understand the world and improve our lot. I find it somewhat amusing (well, frustrating, really) that every new paper coming out of the social or cognitive sciences showing just how limited and biased the human mind is becomes an argument for the irrelevance of rational thinking. As if those discoveries were made in any other way but by deploying the best reasoning abilities we have in order to overcome whatever biases even the researchers involved in those very studies surely suffer from.

To draw on a pertinent analogy: we have incontrovertible evidence that people in general tend to be bad at estimating probabilities, a phenomenon on which the multi-billion dollar casino industry is built. But very few people (other than casino owners, perhaps) would argue that therefore it is useless to teach about the gambler’s fallacy and other pertinent concepts. On the contrary: teaching the rudiments of probability theory is the best way we know of at least partially immunizing fellow human beings from wasting their fortunes at gambling establishments.

The same goes with critical reasoning and open intellectual discourse. They are not a silver bullet, but I guarantee you that once my students are made aware of the standard logical fallacies [8] they see them everywhere (because they are everywhere!), and they are better off for it.

Which brings me to the current project, of which this essay is the beginning and informal “manifesto.” Scientia is a Latin word that means knowledge (and understanding) in the broadest possible terms. It has wider implications than the English term “science,” as it includes natural and social sciences, philosophy, logic, and mathematics, to say the least. It reflects the idea that knowledge draws from multiple sources, some empirical (science), some conceptual (philosophy, math and logic), and it cannot be reduced to or constrained by just one of these sources. Salons, of course, were the social engines of the Age of Reason, and a suitable metaphor for public intellectualism in the 21st century, where the gathering places are more likely to be digital but where discussions can be just as vigorous as those that took place in the rooms made available by Madeleine de Scudéry or the marquise de Rambouillet in 17th century salons.

While I have been thinking for years about a venture like Scientia Salon, and have indeed slowly ratcheted up my involvement in public discourse, first as a scientist and more recently as a philosopher, the final kick in the butt was given to me by my City University of New York (Brooklyn College) colleague Corey Robin. I have never (yet) met Corey, but not long ago I happened across his book, The Reactionary Mind [9], which I found immensely more insightful than much of what has been written of late about why conservatives think the way they do.

More recently, though, I read his short essay in Al Jazeera America, entitled “The responsibility of adjunct intellectuals” [10] and it neatly crystallized a lot of my own unease. Corey points out that academics have always loved to write for other academics using impenetrable jargon (his example of choice is Immanuel Kant), while other thinkers have forever complained about it. He quotes Thomas Hobbes, for instance, as saying that the academic writing of his time was “nothing else … but insignificant trains of strange and barbarous words.”

And yet, observes Robin, we live in an unprecedented era where more and more academics engage openly and vigorously with the public. This, of course, has been made possible by the technologies of the information age, and especially by social networking platforms like blogs, Twitter, Facebook, Google+ and the like. While the average academic article is read by tens or hundreds of people, and it is the rare academic book that reaches 2000 copies, blogs such as my Rationally Speaking (the predecessor to Scientia Salon) is hit by tens, sometimes hundreds of thousands of readers per week, connecting to it from the world over.

Sounds like good news for public intellectualism, no? Well, not exactly, for the simple reason that the tenured academic (such as myself, and Robin) is in decline. While extinction is not an immediate threat, the trend has been as obvious as ominous: in 1971, American universities featured slightly less than 80% full time faculty, complemented by slightly more than 20% adjuncts. In 2009 the two percentages essentially coincided, around 50% per part [11]. It’s not good. Not for adjuncts, not for universities, and not for society at large.

This is not the place to enter into a defense of the tenure system (which, like any social institution, has its pros and cons). But an essential idea behind its inception — which dates back only to the beginning of the 20th century, and was not widely in place until after World War II — is to safeguard faculty from undue administrative and political pressures, giving them relatively free rein as scholars and, you guessed it, public intellectuals. By shifting the balance increasingly toward precariously employed (exploited, really) adjuncts, especially public universities and the States that fund them are effectively undercutting the potential for a new generation of academics interested in engaging in public discourse.

I am not suggesting that the rise of the adjuncts was a premeditated plot by Big Brother to curb the vibrancy of intellectual life — it’s pretty clear that the situation is simply the result of economic decisions coupled with an exceedingly myopic concept of what universities are for. Nor am I claiming that tenured professors are necessarily particularly interested in (or good at) talking to non-peers about what they do. But the fact remains, as Robin so aptly puts it, that “the vast majority of potential public intellectuals do not belong to the academic one percent. They are not forsaking the snappy op-ed for the arcane article. They are not navigating the shoals of publish or perish. They’re grading.”

And this is why I decided to start Scientia Salon and to ask a few colleagues and other interested people to join me. I’m not grading that much compared to the adjuncts working in my Department, and I decided that my time is much better spent working on that “snappy op-ed” (or on essays like this one), which is likely to reach tens of thousands and contribute to the wider debates in our society, rather than on yet another “arcane article” for which I will be lauded by the four or five hyper-specialized colleagues who bothered to read it.

Francis Bacon, arguably the first philosopher of science, famously wrote Ipsa Scientia Potestas Est (which, you may have noticed, is the tag line of Scientia Salon): knowledge is power. While he meant it especially in the sense of harvesting the power of understanding how nature works in order to manipulate her and make human life better, I intend it even more broadly: knowledge and understanding — scientia — of what goes on in the world gives everyone more power over their lives, more ability to influence events, and ultimately more meaning to their existence. This publication aims at making a small contribution in that direction.

Which finally brings me to our manifesto, such as it is:
1) Scientia Salon is a forum for academic and non-academic thinkers who do not shy from the label “public intellectual.”

2) We think intellectualism — in the broader sense of a publicly shared life of the mind — is crucial to the well-being of our society.

3) We acknowledge — as is clear from research in the cognitive sciences — that human beings navigate the world by deploying a complex mixture of reason and emotion, and that they often engage in rationalization more than rationality.

4) Indeed, we think with David Hume that this is a crucial part of human nature, since emotions are necessary in order to actually care about anything in the first place.

5) But we also think that open and reasoned discourse is fundamental for the pursuit of a eudaimonic life on the part of the individual, as well as for the development of a just and democratic society.

6) Scientia, understood as the broadest range of scientific and humanistic disciplines that positively contribute to human understanding, is an essential tool for pursuing that eudaimonic life and achieving that just society.

7) In order to make an impact, we think that writers concerned with these matters ought to aim at a wide audience, avoid unnecessary jargon, and write clearly and engagingly, even humorously when appropriate.

8) We therefore welcome authors and readers who are willing to contribute honestly and substantively to an open dialogue on all matters of the intellect, especially those of general interest to fellow human beings.
Happy writing, reading and commenting, everyone.

Massimo Pigliucci is a biologist and philosopher at the City University of New York. His main interests are in the philosophy of science and pseudoscience. He is the editor-in-chief of Scientia Salon, and his latest book (co-edited with Maarten Boudry) is Philosophy of Pseudoscience: Reconsidering the Demarcation Problem (Chicago Press).

[1] Anti-Intellectualism in American Life, by Richard Hofstadter, 1966.
[2] A Short Course in Intellectual Self Defense, by Normand Baillargeon, 2011.
[3] Necessary Illusions: Thought Control in Democratic Societies, by Noam Chomsky, 1999.
[4] For a good introduction to the context of that quote, and Hume’s moral philosophy in general, see this entry in the Stanford Encyclopedia of Philosophy.
[5] Descartes’ Error: Emotion, Reason, and the Human Brain, by Antonio Damasio, 1994.
[6] Thinking, Fast and Slow, by Daniel Kanheman, 2001.
[7] The Righteous Mind: Why Good People Are Divided by Politics and Religion, by Jonathan Haidt, 2012.
[8] See the exceedingly well done and fun “Thou Shall Not Commit Logical Fallacies” site, and while you are at it, download their handy poster.
[9] The Reactionary Mind: Conservatism from Edmund Burke to Sarah Palin, by Corey Robin, 2011.
[10] The responsibility of adjunct intellectuals, by Corey Robin, Al Jazeera America.
[11] For an in-depth analysis of the “adjuncts phenomenon,” with handy data provided, see “The work of the university” by Stephen Perez and Andrew Litt.

Tom Stafford - The Perspectival Shift: How Experiments on Unconscious Processing Don't Justify the Claims Made for Them

The notion of unconscious processing, perhaps explained best by Daniel Kahneman in Thinking, Fast and Slow (2012), has been used in the field of neuroscience to argue against free will -- see Sam Harris's frustratingly narrow arguments in Free Will (2012).

In this opinion article from Frontiers in Cognitive Science, Tom Stafford argues that:
(1) a widely employed definition of unconscious processing, promoted by John Bargh is incoherent (2) many experiments involve a perspectival sleight of hand taking factors identified from comparison of average group performance and inappropriately ascribing them to the reasoning of individual participants.
In essence, he argues that these studies rely on priming ("an implicit memory effect in which exposure to one stimulus influences a response to another stimulus"), but that unconscious processing is wholly distinct from priming. 

Stafford notes:
John Bargh has influentially defined unconscious processes as those that “do not influence subjective experience in a way that [he or she] can directly detect, understand, or report the occurrence or nature of these events” (Bargh, 1992; Bargh and Morsella, 2008; Huang and Bargh, 2014, p. 14).
But this definition contains a serious reframing of the tradition definition of unconscious from “without awareness of the stimuli” to this newer, broader definition, “without awareness of the influence of the stimuli”. It is this second definition that authors like Sam Harris rely on for their arguments.

This is what Stafford sets out to argue against.

Full Citation:
Stafford T. (2014, Sep 19). The perspectival shift: how experiments on unconscious processing don't justify the claims made for them. Frontiers in Psychology: Cognitive Science; 5:1067. doi: 10.3389/fpsyg.2014.01067

The perspectival shift: how experiments on unconscious processing don't justify the claims made for them

  • Department of Psychology, University of Sheffield, Sheffield, UK

Strong Claims About Unconscious Processing are Unjustified

Recently, there has been widespread focus on studies of unconscious processing that have come out of the field of “social priming” (Doyen et al., 2012; Yong, 2012; Shanks et al., 2013). This focus has primarily been on their replicability (Pashler and Wagenmakers, 2012) and attendant claims of statistical and methodological impropriety (Simmons et al., 2011; Newell and Shanks, 2014). The logic of the claims made has received less attention. In this commentary I draw attention to certain limitations on the inferences which can be drawn about participant's awareness from the experimental methods which are routine in social priming research. Specifically, I ague that (1) a widely employed definition of unconscious processing, promoted by John Bargh is incoherent (2) many experiments involve a perspectival sleight of hand taking factors identified from comparison of average group performance and inappropriately ascribing them to the reasoning of individual participants.

The claims made for the role of unconscious processes are strong. For example, one review states “priming studies have consistently demonstrated that the mere exposure to environmental events is sufficient to directly trigger higher mental processes, in the absence of any conscious intentions or awareness that they operate” (Huang and Bargh, 2014, p. 9). The power of unconscious influences is explicitly placed in opposition to conscious processing “… by logical necessity [priming effects have] reduced the presumed causal role of intentional, conscious processes in higher mental processes” (Bargh and Huang, 2009, p. 128). This leads one review to state “some volitional behavior does not require any conscious awareness at all” (Dijksterhuis and Aarts, 2010, p. 469). Note that the claim is not that unconscious processes are involved in judgment, nor that priming can influence higher mental processes. Rather it is far stronger. Unconscious processes produce judgment, priming triggers higher mental processes, no conscious awareness is required.

I do not wish to question the reality of these priming effects, in that I believe that most of these studies could be replicated. Nor do I deny the challenge they pose to our folk psychology of what influences human behavior (which is often dominated by a simplistic “all acts have deliberate reasons” model). My purpose is merely to draw attention to a disjuncture between the methods used to assess unconscious processes, and the claims made for them in terms of their role in producing action.

Problems with Defining Unconscious by Failure to Report

John Bargh has influentially defined unconscious processes as those that “do not influence subjective experience in a way that [he or she] can directly detect, understand, or report the occurrence or nature of these events” (Bargh, 1992; Bargh and Morsella, 2008; Huang and Bargh, 2014, p. 14). This definition contains a crucial ambiguity. How general must the inability to detect, understand or report be for a process to count as unconscious? Some processes, which we might most appropriately call nonconscious are forever off limits to our introspection (they are “cognitively encapsulated,” Fodor, 1983). Others may not be detected, understood or reported on just one particular occasion. Does this make them unconscious? It seems it does according to the definition promoted by Bargh.

This new definition has been used to support a shift from defining unconscious as “without awareness of the stimuli” to “without awareness of the influence of the stimuli.” This creates two problems. The first problem is it defines the “unconscious” as much by the self-model of the participants as by that of the experimenter. For example, Custers and Aarts (2005) is cited (e.g., by Huang and Bargh, 2014) as an example of subliminal priming which attests to the operation of unconscious goals. The check which was used to ensure that the stimuli really were subliminal was to ask participants at the end of the experiment if they were influenced by the stimuli (Custers and Aarts, 2005, experiment 1). In other words, unconscious operation is defined by participants denying they were influenced. Wilson (2002) has written engagingly about the divergence of our model of our thoughts and feelings from our actual thoughts and feelings. You don't need to be social psychologist to see that there could be many influences which would lead to a participant denying the influence of a stimulus on their choice, and that these might be factors which—while interesting—weaken the claim that this definition of unconscious allows us to focus on processes which are both a natural kind and truly unknown to the subjects (they may, for example, be responding to perceived social pressure to deny the influence of the factors in question).

A highly cited study (Bargh et al., 1996) reported that participants were unconsciously influenced by primes in a scrambled words task to walk more slowly down a corridor upon leaving the experiment. The authors reported, consistent with the definition of unconsciousness that I wish to question, that “no participant believes that the word has an impact on his or her behavior” (Bargh et al., 1996, experiment 1, p. 237). Remarkably, no further test of the awareness of the primes was done on the participants. Instead, a separate 19 participants were tested and funnel debriefed (with half in the experimental condition, so we can expect 9 or 10 to have experienced the elderly primes). The basis for claiming that priming was unconscious is that these participants could not predict what the influence of the primes would be, nor connect them to the elderly stereotype. Aside from issues of statistical power in this check, it seems that no participant was ever directly asked if the primes would affect the specific behavior which was measured. Even if we did ask them, we would have no strong reason to believe that the answers we got were because participants were, in some strong sense, ignorant of the influence of the primes on their behavior. Instead, they may just give answers which fit with common lay beliefs regarding which factors should and shouldn't influence behavior.

This issue of how awareness should be assessed, and of possible biases on subjective reports, is a long-standing one1. Reviews have highlighted the difficulty of demonstrating with certainty that a participant is unaware (Eriksen, 1960; Holender, 1986; Simons et al., 2007; Newell and Shanks, 2014). The way you operationally define consciousness is crucial to whether you can demonstrate perception without it (Reingold and Merikle, 1990; Merikle et al., 2001). In contrast to Bargh et al. (1996) other studies have used stricter methods, such as forced choice questions which remove biases to not report (since they are forced choice) and allow any feelings of awareness (however weak) to inform the choice (see Hannula et al., 2005 for a fuller discussion). It is against this background that Bargh's strategy of defining unconsciousness by failure to report should be judged (Bargh, 1992; Bargh and Morsella, 2008).

The Unjustified Perspectival Shift which Makes Claims about Individual Rationality Based on Group Differences

The second problem introduced by this definition of unconscious concerns how claims of the importance of factors in individual cognition are made from experiments which compare differences in group averages. The logic of many of our behavioral experiments encourages a perspectival shift in which factors which have the major influence on each individual's choices are rendered invisible, while an experimental factor which has a minor influence on each individual's choice is highlighted. This is obviously the intent—the logic of a between subjects design is to pull out the influence of the experimental factor against a background of individual variability. Using this method we identify factors which we can show have a causal influence at the level of group average. It can be a mistake, however, to talk with confidence about the nature of an individual's choice, rather than the average effect over individuals' choices. Consider the statement “Unconscious processes have been shown to produce evaluation and social judgment” (Huang and Bargh, 2014, p. 9). This is simply wrong if we take “produce” to mean “be solely responsible for.” Unconscious processes do not produce, e.g., social judgments. The empirical foundation for this claim is experiments in which social judgment is produced by individuals, who are quite conscious of what they are doing at a macrolevel- i.e., willingly participating in an experiment. Unconscious processes are shown to influence cognitions and behaviors, but they do this as part of the conscious production of these cognitions and behaviors.

If the unconscious nature of these processes is validated at the individual level by asking participants to report what influenced their choices, but then the unconscious process itself is attested to by a difference in group means, it is possible that the experiment identifies a factor which is a minor influence on the choice as a whole. In other words the manipulation can show a strong statistical effect (and we'd hope that as professional experimenters the researchers would design a situation where this was exactly the case), but for a factor which plays a marginal role in each individual's choice. Say the experimental task is to evaluate a word as good or bad. The word is rated as good or bad and each individual, for each judgment, may decide in a way that is consonant with a deliberate and conscious decision making process (i.e., one which is completely at odds with the one being foregrounded by proponents of automatic processing). The dependent variable is reaction time, and the effect of the prime is seen in average differences in reaction time. The influence of the “unconscious” factor may be to speed or slow them in their judgment, while this judgment itself may take a value informed by reasons which the participant is fully aware of. Because “unconscious” effects are manifest this way, it is misleading to talk of the unconscious as “producing” behavior when the only thing tested are differences in characteristics of behavior. This is both because the major element of the behavior may not be affected by the experimental manipulation (e.g., in this case the judgment of the word as good or bad, rather than the speed of the judgment), and because it isn't automatic that an “unconscious” group difference implies an “unconscious” individual judgment.

This perspectival sleight of hand obscures the truly multicausal nature of behavior behind the single controlled cause that is privileged by the experimenter's perspective. Participants in these experiments are, as described, making deliberate and reasoned choices. Their failure to report the influence of the experimental factor may result from an impoverished or incorrect self-model, or it may result merely from the relative unimportance, at an individual level, of the experimental factor in guiding their choices. It is not possible, after all, to report all influences on a behavior, even for a fully informed and rational agent (the “Frame problem,” Dennett, 1978). For these reasons, it is not valid for the conclusion to be drawn that unconscious processes produce behavior, to the extent that this excludes the role of conscious processes in co-producing them. Nor is it valid to infer that unconscious processes significantly determine overall behavior of any individual at any time, as is often implied.

Evidence of differences due to unconscious processes at the group level do nothing to confirm the importance of the unconscious processes in affecting the overall response of each individual. This concern is particularly relevant for studies of unconscious processing when the criteria used to define what is unconscious are based on asking individuals to make judgments about the overall importance of factors. To explore this, consider the tension between experimental effect sizes and wider generalizability.

Larger Effect Sizes Can be in Tension with Generalisability

It is not the case that simple inspection of effect sizes will necessarily reveal the significance of an experimental factor in reasoning. Since effect sizes are based on the amount of variability in a measure, the experimenter typically selects a measure or situation in which variability in minimized. Effect sizes are maximized by situations of tight experimental control—these reduce the influence of non-experimental factors, allowing a purer measure of the experimental manipulation. Note that this means that effect sizes can be uninformative about the importance of the experimental factor in less tightly controlled situations. Indeed, there is a sense in which larger effect size (indicative of tighter experimental control) may actually anti-correlate with generalizability (which requires effects which are robust across situations). One response to failures to replications social priming studies has been that they require some expertise to set up (e.g., Bargh, 2014)—this would seem to be tacit admission of the fragile generalizability of such effects.

Conclusion: Which Influences on Behavior is it Reasonable to Expect a Perfectly Conscious Agent to Report?

The Bargh definition assumes that a rational agent with strong access to the causal mechanisms supporting their decision process could report all factors affecting their decisions. I wish to question this. It would be bizarre if individual agents had access to all the causal factors influencing each of their choices. It would be equally bizarre if they—unaware of the experimenters' interest in a particularly minor factor—were guaranteed to report it at the exact time they were asked. By shifting the defining criteria of unconscious factors to be those which are not reported we open ourselves to the risk that processes which are fully conscious, or potentially conscious, are being used to make claims about the unconscious. This may not be a problem if the revisionist definition of unconscious is born in mind at all times when the implications of these experiments are discussed. Discussion of whether or not this has been the case, both within the scientific literature and in popular discourse, is beyond the scope of this commentary.

The impoverished view of consciousness that results from the Bargh definition is supported by methods which are designed specifically to render conscious deliberation invisible. It remains to be shown that human reasoning is not dominated by self-aware deliberation and based on principles of rationality, which although limited and fallible, can be considered and improved.

Conflict of Interest Statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thanks are due to Robin Scaife, Jules Holroyd and all members of the University of Nottingham, Department of Philosophy reading group on automaticity. Tom Stafford is part-funded by a Leverhulme Trust grant to the University of Nottingham on “Bias and Blame: Do Moral Interactions Modulate the Expression of Implicit Bias?”


1. ^I thank a reviewer for encouraging an exposition on this point.

References at the Frontiers site

Saturday, October 18, 2014

The Pin1 Enzyme and Synaptic Plasticity

This study looked specifically at inhibitory synapses - "The signal it transmits hinders activation of the postsynaptic neuron, making it less likely for it to become activated and emit its action potential,” according to Paola Zacchi, a SISSA [International School for Advanced Studies (Italian: Scuola Internazionale Superiore di Studi Avanzati, SISSA) researcher who coordinated the study.
“When Pin1 is absent from the synapse, signal transmission occurs “at full strength”, but also without control. Instead, when it is present, it regulates signal strength, making it weaker. We observed that Pin1 is able to modify the number of postsynaptic receptors”. The larger the number of receptors capable of binding to the neurotransmitter, the stronger the signal that reaches the postsynaptic membrane. “This also means that Pin1 plays a role in plasticity” explains Zacchi.
The whole article is open access, via Nature Communications, but it's pretty geeky, so we start with a summary of the press release from Neuroscience News, and then below that is most of the original article (minus the methods section which is not relevant to anyone outside the field of neuroscience and molecular biology).

An Enzyme and Synaptic Plasticity

Neuroscience News
October 10, 2014

Study reveals novel role for the Pin1 molecule.

A small, “empty” space teeming with activity: a synapse is a complex structure where the neural signal from the presynaptic neuron, as it travels towards its target turns into a chemical signal capable of crossing the synaptic space before becoming electrical again once on the other side. A synapse is a “dynamic” space not only because of the endless work that goes on there, but also for its ability to change its action over time (synaptic plasticity) as a result of either normal physiological processes (e.g., during learning) or because of disorders due to pathological conditions. A study, mainly carried out by SISSA researchers (which also involved the University of Zurich, LNCIB in Trieste, and EBRI in Rome), showed that a small enzyme (Pin1, peptidyl prolyl isomerase) that plays a mediating role in signal transmission has an effect on synaptic plasticity.

“The synapse we studied is of the inhibitory kind. The signal it transmits hinders activation of the postsynaptic neuron, making it less likely for it to become activated and emit its action potential”, explains Paola Zacchi, a SISSA researcher who coordinated the study. “When Pin1 is absent from the synapse, signal transmission occurs “at full strength”, but also without control. Instead, when it is present, it regulates signal strength, making it weaker. We observed that Pin1 is able to modify the number of postsynaptic receptors”. The larger the number of receptors capable of binding to the neurotransmitter, the stronger the signal that reaches the postsynaptic membrane. “This also means that Pin1 plays a role in plasticity” explains Zacchi.

This image depicts a neuron. Credit SISSA.

More in Detail

How does a synapse work? “A chemical synapse, the most common in vertebrates, is a small gap between nerve cells where the passage of a neural signal occurs”, explains Zacchi. In chemical synapses the two neurons are not in contact but they are separated by a distance of about 20 nanometres. For this reason, the electrical signal travelling along the presynaptic nerve ending is interrupted before resuming on the neuron on the other side of the gap. In between the two nerve cells the electrical signal is translated into a chemical signal (which then becomes electrical again).

“Arrival of the action potential on the presynaptic button causes release, into the interneural space, of molecules of neurotransmitter, which are picked up by receptors on the postsynaptic membrane”, says Zacchi. “If the synapse is excitatory, this leads to postsynaptic activation which, if sufficiently intense, triggers another action potential. If the synapse is inhibitory, as in our studies, the signal suppresses postsynaptic activation and inhibits firing of the electrical potential. In the process of neurotransmitter release and binding, other molecules come into play, such as scaffold proteins, which assemble receptors at the right place on the membrane in front of the neurotransmitter release sites, and neuroligins which act as bridges between the two ends of the synapse as well as interacting with the scaffold proteins. Pin1, the enzyme in the study, interacts with both neuroligins and scaffold proteins.

The Pin1 enzyme has long been known for its role in cancer and the development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s (whereas neuroligins seem to be involved in autism). “Studies like this enhance our understanding of the biochemical mechanisms of synaptic plasticity, extending our knowledge of healthy mechanisms, but also helping those who are trying to understand what can be done in a wide range of pathological conditions”.

Notes about this neuroscience research
Contact: Federica Sgorbissa – SISSA
Source: SISSA press release
Image Source: The image is credited to SISSA and is adapted from the press release
Original Research: Full open access research for “Pin1-dependent signalling negatively affects GABAergic transmission by modulating ​neuroligin2/​gephyrin interaction” by Roberta Antonelli, Rocco Pizzarelli, Andrea Pedroni, Jean-Marc Fritschy, Giannino Del Sal, Enrico Cherubini and Paola Zacchi in Nature Communications; 5 (article #5066). Published online October 9 2014. doi:10.1038/ncomms6066

* * * * *

Pin1-dependent signalling negatively affects GABAergic transmission by modulating ​neuroligin2/​gephyrin interaction

Roberta Antonelli, Rocco Pizzarelli, Andrea Pedroni, Jean-Marc Fritschy, Giannino Del Sal, Enrico Cherubini & Paola Zacchi
Nature Communications 5, Article number (2014, Oct 9): 5066; doi:10.1038/ncomms6066


The cell adhesion molecule ​Neuroligin2 (​NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein ​gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous ​NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis–trans isomerase ​Pin1. This signalling cascade negatively regulates ​NL2’s ability to interact with ​gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of ​NL2, ​gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of ​Pin1-knockout mice (​Pin1−/−) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that ​Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating ​NL2/​gephyrin interaction.


Structural and functional changes of post-synaptic density (PSD) components contribute to regulate synapse formation and plasticity. These remodelling events can affect trafficking, lateral mobility and turnover of several classes of structural and signalling molecules. They often involve interactions among specific proteins regulated by post-translational modifications, such as phosphorylation. At GABAergic synapses, the impact of phosphorylation on the gating properties, surface mobility and trafficking of the gamma-aminobutyric acid A receptors (GABAARs) has been extensively studied1, 2. Much less is known about the effects of phosphorylation of other post-synaptic proteins functionally linked to GABAARs.

An important class of molecules involved in synapse formation, maturation and stabilization comprizes the cell adhesion molecules of the neuroligin (NLs) family3. These post-synaptic proteins functionally coordinate pre and post-synaptic rearrangements by binding, via their extracellular domain, the presynaptically localized neurexins (NRXs) and via specific intracellular motifs, synapse-specific scaffolding molecules4, 5, 6. ​Neuroligin2 (​NL2) isoform is the only known adhesion molecule constitutively present at GABAergic PSDs7, where it drives the recruitment of inhibitory neurotransmitter receptors as well as the scaffolding molecule ​gephyrin6. ​Gephyrin, initially identified as a constituent of purified glycine receptor preparations (GlyR)8, 9, was soon recognized a key player in ​α2 and ​γ2 subunit-containing GABAARs clustering10, 11 and to be a central component of the GABAergic (and glycinergic) PSD8, 12. On the basis of its auto-oligomerization properties, ​gephyrin builds a bidimensional lattice underneath the synaptic membrane, which exposes a high number of binding sites to accumulate GlyR and GABAARs in front of the presynaptic releasing sites13, 14, 15, 16, 17.

NL2 interacts with ​gephyrin through a conserved stretch of amino acid residues highly conserved among all family members6. Site-directed mutagenesis within this binding module identified a specific tyrosine residue (Y770A) whose ​alanine substitution impairs ​NL2 ability to recruit recombinant and endogenous ​gephyrin to post-synaptic sites6. Notably, the corresponding ​tyrosine residue on ​NL1, the isoform enriched at excitatory synapses, was found to be phosphorylated in vivo, preventing ​NL1–​gephyrin interaction while favouring ​PSD95 recruitment at excitatory synapses18. Altogether, these findings point to the existence of intracellular signalling mechanisms able to modulate NL-scaffolding protein interactions by modifying specifically NL properties, leading to alteration in excitatory and inhibitory synaptic transmission.

In the present study, we have investigated whether post-phosphorylation prolyl-isomerization may affect GABAergic transmission in a similar manner. This signalling cascade targets ​serine and ​threonine residues preceding a ​proline residue to promote conformational changes on its substrate19. This effect is achieved by a unique enzyme, peptidyl-prolyl isomerase ​Pin1, whose catalytic activity facilitates the cis–trans isomerization of the peptide bond20, 21. Notably, ​Pin1 was found to interact with ​gephyrin and to alter its overall conformation, thus enhancing its ability to bind the GlyR22.
Here, we provide evidence that endogenous ​NL2 can be phosphorylated at its unique ​Pin1 consensus motif thus rendering it able to physically recruit the phospho-specific effector ​Pin1. We show that post-phosphorylation prolyl-isomerization can regulate ​NL2’s ability to complex with ​gephyrin. Specifically, ​Pin1-mediated propyl-isomerization of phosphorylated serine 714 negatively modulates ​NL2–​gephyrin complex formation, down-regulating GABAergic synaptic transmission.


Endogenous ​NL2 undergoes proline-directed phosphorylation

The cytoplasmic domain (CD) of ​NL2 possesses a unique consensus motif for proline-directed phosphorylation, S714-P, located 15 amino acids apart from the transmembrane domain (Fig. 1a). To assess whether this site can undergo phosphorylation in vivo we used the mitotic phosphoprotein monoclonal 2 (MPM2) antibody that specifically recognizes phosphorylated S/T-P motifs (Davis et al.23). Endogenous ​NL2 was therefore immunoprecipitated from mouse brain homogenates using an affinity-purified polyclonal antibody raised against its CD or normal mouse IgG as negative control. Western blotting using the MPM2 antibody revealed a band at around 120 kDa that corresponds to the upper band of the doublet recognized by the ​NL2 antibody in parallel immunoprecipitation experiments (Fig. 1b), suggesting that at least a fraction of ​NL2 can be phosphorylated at its unique ​Pin1 consensus motif. To demonstrate that phosphorylation at serine 714 is the event responsible for ​NL2 detection by the MPM2 antibody, we generated the phospho-defective point mutant ​NL2HA-S714A. This mutation was introduced into a ​NL2HA hampered in ​gephyrin binding (​NL2HA-S714A-Δgephyrin-binding domain, GBD) (see Supplementary Fig. 1), to exclude the possibility that the MPM2 antibody would immune-react with phosphorylated ​Pin1 consensus motifs on endogenous ​gephyrin, which is, at the same time, a ​Pin1 target22 and an interacting partner of ​NL2 (ref. 6). Under these conditions, the MPM2 antibody efficiently immunoprecipitated only ​NL2HA-ΔGBD but not the corresponding point mutant, as indicated by the anti-HA immunoblot (Fig. 1c), thus demonstrating that S714 can be found phosphorylated on ​NL2.
Figure 1: ​NL2 is a proline-directed substrate. 
NL2 is a proline-directed substrate.
(a) Amino acid sequence of the ​NL2 CD. In bold is marked the unique ​Pin1 consensus motif (S714-P). The gephyrin-binding domain and the proline-rich region are highlighted in bold. (b) Representative immunoblotting of endogenous ​NL2 immunoprecipitated (IP) from mouse brain and probed with the anti-MPM2 that specifically recognizes phosphorylated S/T-P motifs and anti-​NL2. Rabbit IgGs were used as negative control (IgG) (n=4). (c) Representative immunoblotting of overexpressed ​NL2HA lacking the ​gephyrin binding domain (​NL2HA-ΔGBD) and the corresponding point mutant (​NL2HA-ΔGBDSer714Ala) immunoprecipitated by the phospho-specific MPM2 antibody. Western blot analysis was carried out with anti-HA monoclonal antibody. Mouse IgGs were used as negative control (n=5). (d) Co-immunoprecipitation (Co-IP) of endogenous ​NL2 and ​Pin1 from DSP cross-linked brain homogenates of ​Pin1+/+ or ​Pin1−/− mice. Western blots were performed with anti-​NL2 polyclonal and anti-​Pin1 monoclonal antibodies. Mouse IgGs were used as negative control. Asterisk indicate the IgG light chains (n=6). (e) FLAG epitopes from cross-linked samples of HEK293 cells co-expressing ​Pin1-​FLAG and ​NL2HA-ΔGBD or ​NL2HA-ΔGBDS714 were immunoprecipitated by anti-FLAG antibody. Western blot was performed with anti-HA and anti-FLAG monoclonal antibodies. Mouse IgGs were used as negative control (n=4). Full images of western blots are in Supplementary Fig. 5.
The essential feature of proline-directed phosphorylation as a signalling mechanism relies on the ability of phosphorylated S/T-P motifs to recruit the prolyl isomerase ​Pin1 (refs 19, 24). To test whether this unique phospho-epitope is able to recruit the effector molecule of the signalling cascade, we performed co-immunoprecipitation experiments from ​Pin1+/+ and ​Pin1−/− brain lysates. This approach unveiled that ​Pin1 can be detected in ​NL2, but not in control, immunoprecipitates or in the absence of ​Pin1 expression (Fig. 1d). To exclude the possibility that ​Pin1 co-precipitated by ​NL2 is bound to endogenous ​gephyrin, these assays were performed on co-expression of ​NL2HA-ΔGBD and ​Pin1-​FLAG in HEK293 cells. Cell lysates were immunoprecipitated with the anti-FLAG antibody and bound protein complexes analysed by western blotting using anti-HA and anti-FLAG antibodies for ​NL2 and ​Pin1 detection, respectively. As shown in Fig. 1e, while ​NL2HA-ΔGBD was still able to be immunoprecipitated from cells expressing ​Pin1-​FLAG, S714 to ​alanine mutagenesis completely abolished such interaction, indicating that S714 represents a newly identified ​Pin1 target.
Pin1 modulates ​gephyrin–​NL2 interaction

The observation that two fundamental components of the GABAergic PSD are both targets of proline-directed phosphorylation prompted us to investigate whether such signalling cascade would modulate their interaction. To this end, we initially co-expressed ​gephyrin-​FLAG and ​NL2HA in HEK293 cells and examined the amount of ​NL2HA that complex with ​gephyrin-​FLAG at 48 h after treating the cells with the selective and reversible inhibitor of ​Pin1 isomerase activity ​PiB (IC50 of approximately 1.5 μM) (ref. 25). As shown in Fig. 2a, even though the anti-FLAG antibody immunoprecipitated comparable amounts of ​gephyrin-​FLAG, a significant increase (64%) in the amount of co-precipitated ​NL2HA was observed on ​PiB treatment as compared with mock-treated cells (​dimethylsulfoxide, ​DMSO). Interestingly, a marked increase (140%) was detected on ​gephyrin-​FLAG co-precipitation by ​NL2HA-S714A as compared with ​NL2HA, indicating that ​Pin1 exerts a negative control on ​NL2–​gephyrin complex formation, at least in part, through ​NL2 prolyl-isomerization (Fig. 2b).
Figure 2: ​Pin1 negatively modulates ​NL2/​gephyrin interaction.
Pin1 negatively modulates NL2/gephyrin interaction.
(a) Representative IP of FLAG epitopes from samples of HEK293 cells co-expressing ​gephyrin-​FLAG and ​NL2HA and treated for 48 h with ​PiB 2.5 μM, ​DMSO (mock) or untreated. IP was also performed on ​NL2HA single transfected cells as a negative control. Nitrocellulose membranes were probed with anti-HA and anti-FLAG antibodies. The histogram on the right shows the relative amount of ​NL2 co-precipitated by ​gephyrin-​FLAG in control and ​PiB treated cells obtained from densitometric analysis (n=5, mean values±s.d., **P<0.001, Student’s t-test). (b) Lysates of HEK cells transfected with ​gephyrin-​FLAG in the presence of ​NL2HA or ​NL2HA-S714A or with ​gephyrin alone (as a negative control) were immunoprecipitated with anti-HA agarose. Immunoprecipitates were analysed by western blotting using anti-FLAG and anti-HA monoclonal antibodies. Arrowhead indicates the IgG heavy chains. The histogram on the right shows the relative amount of ​gephyrin-​FLAG in complex with either ​NL2HA or ​NL2HA-S714A co-precipitated by anti-HA agarose obtained from densitometric analysis (n=5, mean values±s.d., **P<0.001, Student’s t-test). (c) Co-IP of endogenous ​NL2/​gephyrin complexes from DSP cross-linked brain homogenates of ​Pin1+/+ or ​Pin1−/− mice. Western blots were performed with anti-​NL2 polyclonal and anti-​gephyrin monoclonal antibodies. Rabbit IgGs were used as negative control. An increased amount of ​gephyrin co-precipitates in complex with ​NL2 in the absence of ​Pin1 expression. Arrowhead indicates the IgG heavy chains. The histogram on the right shows the relative amount (obtained from densitometric analysis) of endogenous ​gephyrin co-precipitated by endogenous ​NL2 from both mouse genotypes (n=8, mean values±s.d., *P<0.01, Student’s t-test). (d) A similar experiment described in c was carries out on hippocampus isolated from of ​Pin1+/+ or ​Pin1−/− mice. The histogram on the right shows the relative amount (obtained from densitometric analysis) of endogenous ​gephyrin co-precipitated by endogenous ​NL2 from both mouse genotypes (n=4, mean values±s.d., **P<0.001, Student’s t-test). Full images of western blots are in Supplementary Fig. 5.
This issue was then investigated using a source of native ​NL2–​gephyrin complexes mouse brain homogenates from both genotypes. For these experiments, endogenous ​NL2 was immunoprecipitated using a rabbit polyclonal anti-​NL2 antibody and the co-precipitated ​gephyrin fraction was visualized by the monoclonal 3B11 antibody (Fig. 2c). In the absence of ​Pin1 expression, the amount of ​gephyrin co-precipitated by ​NL2 was increased by 40% as compared with ​Pin1 expressing neurons. This approach was also applied on hippocampal tissues isolated from both mouse genotypes. Here, the enrichment of ​gephyrin co-precipitated by ​NL2 in the absence of ​Pin1 expression was even more dramatic as compared with the amount detected from whole brain (130% increase; Fig. 2d), suggesting a strong impact of such signalling pathway on GABAergic synapses of the hippocampus.

Characterization of ​gephyrinPin1 sites S270-P and S319-P

The scaffolding molecule ​gephyrin possesses 10 putative ​Pin1 consensus motifs, the majority of them being concentrated in the central region (C-domain)26. To determine whether specific ​Pin1 sites may contribute to enhance ​NL2/​gephyrin complex formation, we decided to focus on those located close to, or within, the ​NL2 binding site on ​gephyrin. A previous yeast two-hybrid screening identified a large portion of ​gephyrin encompassing the E-domain and part of the C-domain as the region involved in ​NL2 interaction6. We re-examine this issue by generating eGFP-tagged ​gephyrin truncated version to be tested in GST-​NL2-CD pulldown assays. HEK293 cells transfected with different eGFP-​gephyrin variants were incubated with GST-​NL2-CD loaded beads or with GST alone as negative controls. As shown in Fig. 3a, while ​gephyrin 310–736 was recruited even better than the wild-type (WT) version, the mutants ​gephyrin 326–736 and ​gephyrin 1–310 (​gephyrin GC) displayed a reduced binding activity as compared to both ​gephyrin full-length (FL) and the truncated version 310–736 (Fig. 3a). Since the two E-domain ​gephyrin versions, showing such a striking difference in the binding affinity, differ only for a short stretch of amino acids, we generated the deletion mutant removing, from the FL protein, only the residues contained in this region but belonging to the E-domain itself (​gephyrinΔ319–329) and assayed it for ​NL2 binding. Interestingly, the lack of this short sequence almost completely abolished the interaction of ​gephyrin with ​NL2 (Fig. 3b), indicating that epitope(s) contained in the C-domain together with this minimal binding module are involved in ​gephyrin recruitment.
Figure 3: Impact of ​gephyrin S270A and S319A in ​NL2/​gephyrin interaction. 
Impact of gephyrin S270A and S319A in NL2/gephyrin interaction.
(a) GST-​NL2-CD pulldown from samples of HEK293 expressing EGFP-​gephyrin full-length (FL), EGFP-​gephyrin 310–736 (E-310), EGFP-​gephyrin 326–736 (E-326) and EGFP-​gephyrin GC. GST was used as negative control. Pulled down eGFP​gephyrin variants were detected using an anti-GFP monoclonal antibody. The bottom panels show the levels of GST and GST-​NL2-CD in the pulldown assays (Ponceau staining) (n=8). (b) EGFP-​gephyrin Δ319 to 329 was tested in similar pulldown assays. Western blots in a and b were performed using anti-GFP antibody. ​Gephyrin requires amino acid sequence 319–329 for its efficient recruitment by ​NL2 (n=6). (c) Representative IP of HA epitopes from samples of HEK293 cells co-expressing ​NL2HA and EGFP-​gephyrin WT, EGFP-​gephyrinS270A or EGFP-​gephyrinS319A. Nitrocellulose membranes were probed with anti-HA and anti-GFP antibodies. EGFP-​gephyrin single transfected cells incubated with HA agarose were used as negative controls. The histogram on the right shows the relative amount of eGFP-gephyrinWT and point mutants co-precipitated by ​NL2HA (n=4, mean values±s.d., P>0.05). (d) Representative images of hippocampal neurons transfected with EGFP-​gephyrin and EGFP-​gephyrinS270A point mutant immunolabeled for endogenous ​NL2 (magenta) and ​VGAT (blue) at DIV10. Enlarged boxed areas are shown aside to the corresponding full view image. Post-synaptic clustering is demonstrated by apposition of ​gephyrin/​NL2 clusters to ​VGAT positive terminals on the merge window. Scale bars, 20 μm in full view images and 5 μm in enlarged panels. (e) Distribution histograms of the % of ​gephyrin clusters colabeled with ​NL2 (79±5% in EGFP-​gephyrinWT versus 77±4% in EGFP-​gephyrinS270A), % of ​NL2 clusters colabeled with ​gephyrin (48±5% in EGFP-​gephyrinWT versus 71±4% in EGFP-​gephyrinS270A), % of ​NL2 synaptically localized (29±2% in EGFP-​gephyrinWT versus 43±6% in EGFP-​gephyrinS270A) and ​NL2 clusters intensity (119±15 a.u. in EGFP-​gephyrinWT versus 102 a.u.±6 in EGFP-​gephyrinS270A). The number of transfected hippocampal neurons investigated in each experiments (four independent experiments) were as follow: n=15 for eGFP-​gephyrinWT, n=10 for eGFP-gepyrinS270A (for each neurons at least 4 dendritic regions of interests were measured, mean values±s.d., *P<0.01, Student’s t-test).
On the basis of these results, two ​Pin1 consensus sites were further characterized, namely S319-P, located at the edge of the minimal binding module, and S270-P, positioned in its proximity, still contained, in the C-domain participating in ​NL2 binding. To this end, we introduced point mutations in eGFP-​gephyrin to create S319A and S270A mutants and tested them for their ability to interact with ​NL2HA. As judged by co-immunoprecipitation experiments, no significant differences were observed in binding capacity of the mutants as compared with ​gephyrin WT (Fig. 3c). These constructs were also overexpressed in cultured hippocampal neurons to analyse and quantify their impact on endogenous ​NL2 distribution using immunofluorescence staining and confocal microscopy. As previously reported, neurons expressing the S270A mutants had an increased number, unchanged in size, of ​gephyrin clusters compared with eGFP-​gephyrin WT27 (18.9±1.7 per 20 μm dendritic segment versus 6.5±0.6, P=0.00015). The expression of the S319A construct produced a dramatic decrease in cluster density associated with a diffuse cytoplasmic staining. This latter effect seems to correlate with the intrinsic instability of the mutant protein that undergoes a high rate of degradation on neuronal expression (data not shown), hampering its further characterization. Clusters formed by ​gephyrin S270A co-localized with ​NL2 at the same extent as the WT protein (around 78%; Fig. 3d,e). The fraction of ​NL2 clusters co-localizing with S270A mutant as well as their synaptic localization were increased as compared with ​gephyrin WT but their intensity values (calculated by normalizing cluster fluorescence intensity to cluster area and expressed in a.u.: 119 a.u.±15.2 versus 102±6.3) were unchanged (Fig. 3e). These data indicate that the increase in ​NL2/​gephyrin S270 interaction observed by immunoprecipitation is simply due to the augmented S270A cluster density and not to an enhance affinity of the mutant for ​NL2.

Pin1 selectively controls ​NL2 synaptic enrichment
Pin1 has emerged as a negative regulator of ​gephyrin–​NL2 interaction. Since these protein complexes are mainly localized at the plasma membrane, we tested whether ​Pin1 affects the amount of ​NL2 transported to, or maintained at, the neuronal plasma membrane. To this end, cultured hippocampal neurons derived from ​Pin1+/+ and ​Pin1−/− mice were subjected to surface biotinylation assay. Cell surface proteins were treated with the membrane-impermeant ​sulfo-NHS-biotin reagent, then isolated by binding to Streptavidin beads and probed with anti-​NL2 antibody. To check for unspecific protein binding during surface biotinylation experiments, hippocampal neurons not labelled with ​biotin were processed with biotinylated samples. Western blot detecting the intracellular glycophosphatidylinositol-anchored protein Flotilin1 was included to ensure that similar amount of associated membrane proteins, biotinylated or not, where incubated with Streptavidin beads. No major differences on the total content of membrane localized ​NL2 were observed between ​Pin1+/+ and ​Pin1−/− (Fig. 4a).
Figure 4: ​Pin1 enhances ​NL2 synaptic content not its surface abundance. 
Pin1 enhances NL2 synaptic content not its surface abundance.
(a) Surface ​NL2 derived from cultured hippocampal neurons of ​Pin1+/+ and ​Pin1−/− mice was isolated by biotinylation assay and detected by anti-​NL2 antibody. No biotinylated neuronal cells were processed in parallel to evaluate unspecific ​NL2 binding. Western blot detecting glycophosphatidylinositol-anchored Flotilin was used as loading control (n=4). Full images of western blots are in Supplementary Fig. 5. (b) Typical examples of hippocampal neurons from ​Pin1+/+ and ​Pin1−/− immunolabeled for endogenous ​gephyrin (magenta), ​NL2 (green) and ​VGAT (blue) at DIV10. Enlarged boxed areas are shown aside to the corresponding full view image. Post-synaptic clustering is demonstrated by apposition of ​gephyrin/​NL2 clusters to ​VGAT positive terminals on the merge window. Scale bars, 20 μm in full view images and 5 μm in enlarged panels. (c) Distribution histograms of ​NL2 cluster density (normalized to 100 μm2), the average cluster size and intensity in ​Pin1+/+ and ​Pin1−/− hippocampal neurons. (d) Distribution histograms of the percentage of ​NL2 co-localizing with gephryin and the percentage of double labelled ​NL2/​gephyrin puncta overlapping with the presynaptic marker ​VGAT. (e) Distribution histograms of ​gephyrin cluster density (normalized to 100 μm2), the average cluster size and intensity (calculated as described in c) in both mouse genotypes. The number of hippocampal neurons investigated in each experiments (three independent experiments) were as follows: n=10 for ​Pin1+/+, n=12 for ​Pin1−/−. For each neurons, at least five dendritic regions of interests were measured, mean values±s.d., **P<0.001, ***P<0.0001, Student’s t-test).
These results allow excluding the involvement of ​Pin1 in ​NL2 transport and/or turnover at the plasma membrane. Surface biotinylation represents an experimental approach that cannot provide an accurate analysis of protein distributions among different membrane domains. Since ​NL2 is enriched at GABAergic synapses, but is also distributed on extrasynaptic sites28, with this approach differences in ​NL2 partitioning between these two compartments might have been missed.

To this aim, immunocytochemical experiments were performed in dissociated ​Pin1+/+ and ​Pin1−/− hippocampal neurons co-labelled for ​NL2, ​gephyrin and ​VGAT, a specific marker of GABAergic innervations29 (Fig. 4b). In the absence of ​Pin1 expression a significant increase in ​NL2 cluster size (2.4 μm2±0.2 versus 1.7 μm2±0.2, P=0.00044) and intensity (92 a.u.±4.0 versus 58 a.u.±2, P<0.00048) was observed as compared with WT neurons, while no major changes in ​NL2 cluster density were detected (Fig. 4c). The fraction of ​NL2-positive clusters co-localized with endogenous ​gephyrin puncta was also enhanced in ​Pin1−/− cells (80±3.0% versus 60±5%, P=0.00013) and found enriched at post-synaptic sites, as demonstrated by the higher percentage of ​NL2/​gephyrin co-stained puncta overlapping with the presynaptic marker ​VGAT (48±4% versus 33±4%, P=0.0008; Fig. 4d). ​Gephyrin puncta appeared slightly, but significantly, increased in size while their density and intensity values were unchanged as compared with ​Pin1+/+ (Fig. 4e). These observations suggest that the absence of ​Pin1 promotes the formation and/or stabilization of ​NL2/​gephyrin complexes at GABAergic post-synaptic sites.

NL2/​gephyrin complex modulates synaptic abundance of GABAARs

The recruitment of GABAARs at synaptic sites is functionally coupled to NLs expression levels as well as to the ​gephyrin scaffold6. To assess whether the enhanced ​NL2/​gephyrin complex formation detected at GABAergic synapses similarly affects the distribution of synaptic ​γ2 subunit-containing GABAARs, we performed a quantitative evaluation of the ​γ2 subunit present in synaptosome suspensions isolated from the hippocampus of ​Pin1+/+ and ​Pin1−/− mice. Quantitative immunoblot analysis was also extended to ​NL2 and ​gephyrin to further verify their synaptic enrichment. As shown in Fig. 5a, the amount of all three markers investigated was significant increased in ​Pin1−/− mice as compared with ​Pin1+/+. The synaptic enrichment (synaptic fraction versus homogenate) was 35±5% for the ​NL2, 30±6% for the ​γ2 subunit and 20±4% for ​gephyrin.
Figure 5: Synaptic enrichment of GABAARs is achieved in ​Pin1−/−. 
Synaptic enrichment of GABAARs is achieved in Pin1−/−.
(a) Representative immunoblots of ​NL2, ​gephyrin and ​γ2 subunit of GABAA receptor extracted from the hippocampus of ​Pin1+/+ and ​Pin1−/− mice (littermates) in two different sets of experiments. Total proteins from the homogenates and synaptosome suspension fractions were analysed by western blotting. Below: quantification of the indicated antigens extracted from hippocampal tissues of ​Pin1+/+ and ​Pin1−/− mice. All markers analysed are enriched at inhibitory synapses. Western blot to actin was done as loading control. ​Pin1 immunoblot indicates hyppocampus from ​Pin1+/+ and ​Pin1−/− (n=6 littermate pairs, mean values±s.d, *P<0.05, Student’s t-test) Full images of western blots are in Supplementary Fig. 5. (b) Representative confocal micrographs of frontal brain sections showing segments of the SR and SO of the CA1 region of the hippocampus from adult ​Pin1+/+ and ​Pin1−/− mice immunolabeled for ​gephyrin (magenta) and ​VGAT (green). Scale bar, 5 μm. (c) Quantification of ​gephyrin punctum density (normalized to 100 μm2) and their percentage of colocalization with the presynaptic marker ​VGAT in both mouse genotypes. (d) Confocal micrographs as in a immunolabeled for ​GABAA receptor γ2 subunit (green) and ​VGAT (magenta). (e) Quantification of ​γ2 subunit punctum and their percentage of colocalization with ​VGAT in both mouse genotypes. The number of ​gephyrin, ​γ2, ​gephyrin and ​VGAT puncta was assessed in at least eight sections for each genotypes (​Pin1+/+ and ​Pin1−/−), by taking at least four images of SR and SO of the CA1 region of each hippocampus in each set of experiments (n=3). Mean values±s.d., *P<0.05, Student’s t-test. Scale bar, 5 μm.
We also examined the number of puncta labelled for ​gephyrin and ​γ2 subunit-specific antibodies, as well as their levels of colocalization with the presynaptic marker ​VGAT, in the CA1 region of the hippocampus of both genotypes. The staining pattern of ​gephyrin in ​Pin1−/− demonstrated a slight increase in the number of clusters both in the stratum oriens (SO) and stratum radiatum (SR) as compared with ​Pin1+/+ (SO 16±3 clusters per 100 μm2 and SR 28±3 clusters per 100 μm2 versus SO 10±2 clusters per 100 μm2 and SR 19±3 clusters per 100 μm2; P<0.05; Fig. 5b,c). This increase was paralleled by a small increase (around 6–8%) in ​gephyrin puncta co-localized with presynaptic ​VGAT (SO 30±2% and SR 39±1.4% versus SO 24±2% and SR 31±2%; P<0.05; Fig. 5b,c). The average cluster size and intensity were similar in both genotypes (3.6 μm2±0.2 versus 3.5 μm2±0.3 and 61±7 versus 65±4 a.u. for cluster size and intensity in ​Pin1−/− versus ​Pin1+/+, respectively).

The ​γ2 subunit staining pattern exhibited a similar cluster density in the two strata analysed in both genotypes (SO 8±2 and SR 18±2 versus SO 8±1 and SR 17±1.2; P>0.05; Fig. 5d,e). A small, although significant, increase in their intensity was evident (120±3 RFU versus 106±2 RFU in ​Pin1−/− versus ​Pin1+/+; P<0.05) but they were similar in size (4.3 μm2±0.5 versus 3.7 μm2±0.5). ​VGAT colocalization was increased by 10–15% in tissue from knockout animals (SO 38.9±2.7% and SR 52±3% versus SO 29±2% and SR 36±3%; P<0.05; Fig. 5d,e). The changes in ​gephyrin and ​γ2 subunit synaptic fraction are not due to an increase in synapses numbers, the density of inhibitory terminals being unaltered between the two genotypes, as assessed by quantification of ​VGAT immunolabeling (SO 14±2% and SR 22±3% versus SO 13±2% and SR 21±3%; P>0.05).
Altogether, these data indicate that the enhanced interaction between ​gephyrin and ​NL2 observed in the absence of ​Pin1 is associated with a concomitant increase in the synaptic recruitment of ​γ2 subunit-containing GABAARs.

Pin1 signalling affects the number of synaptic GABAARs

To functionally explore whether the enrichment of ​γ2 subunit-containing GABAARs in ​Pin1−/− mice affects GABAergic transmission, whole-cell recordings in voltage clamp configuration were performed from CA1 principal cells in hippocampal slices obtained from ​Pin1+/+ and ​Pin1−/− mice at postnatal (P) day P10–P13. These neurons presented similar resting membrane potential (Vrest) and input resistance (Rin) values (data not shown), thus indicating that ​Pin1 does not affect the passive membrane properties of principal cells. Spontaneous GABAA-mediated inhibitory post-synaptic currents (sIPSCs) were then recorded from both genotypes in the presence of ​6,7-dinitroquinoxaline-2,3-dione (​DNQX; 20 μM) to block AMPA-mediated excitatory post-synaptic currents (sEPSCs). As shown in Fig. 6a, recordings from ​Pin1−/− mice exhibited sIPSCs of higher amplitude values compared with control littermates (106±12 pA versus 62±8 pA; P<0.05), in the absence of any significant change in frequency (4.2±0.5 Hz versus 3.6±0.6 Hz; P>0.05; Fig. 6b). The amplitude distribution histogram of sIPSCs recorded in ​Pin1−/− unveiled a clear peak at ~200 pA (Fig. 6c). The observed effects were selective for sIPSCs since no significant differences in amplitude (22±2 pAin ​Pin1−/− mice and 27±4 pA in ​Pin1+/+; n=6 for both genotypes; P>0.05) or frequency (1.7±0.3 Hz in ​Pin1−/− mice and 1.3±0.4 Hz in ​Pin1+/+ mice; P>0.05) of sEPSCs (recorded in the presence of picrotoxin, PTX, 100 μM) were detected between the two genotypes (Supplementary Fig. 2a,b).
Figure 6: ​Pin1 affects the amplitude but not the frequency of sIPSCs.
Pin1 affects the amplitude but not the frequency of sIPSCs.
(a) Representative traces of sIPSCs recorded from CA1 principal cells at P11 in hippocampal slices from ​Pin1+/+ (black) and ​Pin1−/− mice (grey). Note higher amplitude events in ​Pin1−/− mice. (b) Each column represents the mean frequency and amplitude values of sIPSCs recorded from ​Pin1+/+ (black, n=9) and Pin−/− mice (grey, n=8). *P<0.05, Student’s t-test). (c) Amplitude distribution histograms of sIPSCs recorded in ​Pin1+/+ (1,030 events; black) and in ​Pin1−/− mice (1,412 events; grey). Note the appearance of a clear peak at ~200 pA in ​Pin1−/− mice.
Spontaneous inhibitory events from hippocampal neurons in culture overexpressing the ​NL2HA-S714A mutation exhibited, compared with ​NL2HA-transfected cells, a significant increase in amplitude (but not frequency), which in part mimicked the phenotype observe in ​Pin1−/− mice, suggesting that the interaction of ​Pin1 with ​NL2 is critical for this effect (Fig. 7a). As shown in the cumulative amplitude plot (Fig. 7b), the curve obtained from ​NL2HA-S714A transfected cells was shifted to the right as compared with cells expressing ​NL2HA (P<0.05).
Figure 7: Changes in amplitude of sIPSCs involve the interaction of ​Pin1 with ​NL2.
Changes in amplitude of sIPSCs involve the interaction of Pin1 with NL2.
(a) Samples traces of sIPSCs recorded from hippocampal neurons in culture expressing either the ​NL2HA or the ​NL2HA-S714A mutation. (b) Amplitude and inter-event interval (IEI) plots of sIPSCs recorded in cells transfected either with the ​NL2HA (black; n=7) or the ​NL2HA-S714A point mutant (grey; n=12). P<0.05; Kolmogorov–Smirnov test. Note the shift to the right of the cumulative amplitude distribution curve obtained from cells transfected with the mutant as compared to controls.
The selective increase in amplitude of sIPSCs detected in ​Pin1−/− mice suggest a post-synaptic site of action. This may involve an increase in the number of active GABAARs or changes in single-receptor channel conductance. To distinguish between these two possibilities, peak-scaled non-stationary fluctuations analysis of sIPSCs was performed only on stable recordings with no time-dependent changes in either peak amplitude, 10–90% rise time and decay time (Fig. 8a) (electrotonic filtering was excluded on the basis of no correlation between 10–90% rise time and decay time30). Plotting the mean current amplitude versus variance and fitting individual points with the parabolic equation (equation (2) in the methods; Fig. 8b), allowed estimating single-channel conductance and the number of channels open at the peak of spontaneous IPSCs. The single-channel conductance was calculated according to equation (3), assuming a reversal potential for chloride equal to 0. Interestingly, while the values of single-channel conductance were similar in both genotypes (Fig. 8c), the average number of active channels open at the peak of sIPSCs (Np) was significantly increased in ​Pin1−/− mice compared with controls (53±11 versus 26±5; P=0.03; Fig. 8c).
Figure 8: ​Pin1 controls the number of active receptor channels at GABAergic synapses. 
Pin1 controls the number of active receptor channels at GABAergic synapses.
(a) Individual sIPSCs from ​Pin1+/+ (black) and ​Pin1−/− mice (grey) are shown with the average currents (thick lines). (b) Current/variance relationships for sIPSCs shown in a (c) Summary plots of weighted mean channel conductance (43±3 pS and 43±3 pS, P=0.9, Student’s t-test) and number of GABAA receptor channels (Np) in wt (black; n=8) and in ​Pin1−/− mice (gray; n=5). *P=0.03, Student’s t-test.
To further evaluate the possibility that higher amplitude inhibitory events recorded in ​Pin1−/− mice may originate from GABAARs containing different subunits, we measured in both genotypes the decay time constants of small and large amplitude events. Spontaneous IPSCs were plotted against their decay half-widths and arbitrarily divided in two main classes whose amplitude was <or>150 pA (Fig. 9a, in green and blue, respectively). Notably, larger amplitude events (>150 pA) prevailed in ​Pin1−/− mice. No differences in decay of sIPSCs <or>150 pA were observed between ​Pin1+/+ and ​Pin1−/− mice, thus excluding the involvement of multiple receptor subtypes with different kinetics (the 90–10% decay (τ) of sIPSCs <150 pA was 9±1 ms in Pin−/− mice and 11±2 ms in ​Pin1+/+; P>0.05; τ of sIPSCs >150 pA was 11±2 ms in Pin−/− mice and 10±2 ms in ​Pin1+/+; P>0.05. The 90–10% decay time (τ90–10%) of all sIPSCs was 11±2 ms and 10±2 ms in ​Pin1+/+ and ​Pin1−/− mice, respectively; Fig. 9b,c, P>0.05). These data altogether suggest that the observed increase in amplitude of sIPSCs in ​Pin1−/− mice is exclusively due a genuine increase in number of GABAARs composed of the same subunits.
Figure 9: ​Pin1 does not affect the decay kinetics of spontaneous IPSCs. 
Pin1 does not affect the decay kinetics of spontaneous IPSCs.
(a) The peak amplitude of individual sIPSCs <150 pA (green) and >150 pA (blue) is plotted against their decay half-widths (τ50%) in ​Pin1+/+ and in ​Pin1−/− mice. (b) In the upper part, average traces of spontaneous IPSCs shown in a. In the lower part, average traces are normalized and superimposed. (c) Each column represents the mean 90–10% decay time constant of spontaneous IPSCs in ​Pin1+/+ and ​Pin1−/− mice, <150 pA (green), n=8 and 7, respectively and >150 pA (blue), n=6 and 7, respectively. For all comparisons, P>0.05, Student’s t-test.
GABA release and tonic inhibition are unaltered in ​Pin1−/−

In a previous study, we demonstrated that the functional knockdown of ​NL2 was accompanied by a reduction in the probability of ​GABA release31, thus underlying the role of NLs as retrograde regulators of presynaptic function. Therefore, we evaluated here whether ​Pin1-dependent modulation of ​NL2–​gephyrin interaction could also affect ​GABA release from presynaptic nerve terminals. To this end, we used ​1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid (​TPMPA), a low affinity competitive GABAAR antagonist32. This approach allowed to compare differences in presynaptic ​GABA transients between ​Pin1+/+ and ​Pin1−/− mice. Similar reduction of sIPSCs amplitude in both genotypes (51±6% versus 54±8%, P>0.05, Supplementary Fig. 3a,b) was detected on bath application of ​TPMPA (200 μM), thus excluding a transsynaptic action of ​Pin1 on ​GABA release.

Part of ​GABA released during synaptic activity may escape the cleft and invade the extracellular space to activate extrasynaptic high affinity GABAARs. This feature generates a persistent GABAA-mediated conductance33 that is involved in a number of physiological processes34. To determine whether ​Pin1 signalling affects extrasynaptic GABAARs, we analysed the tonic GABAA-mediated conductance in both ​Pin1+/+ and ​Pin1−/−mice. The tonic conductance was assessed by the shift of the holding current induced by application of the GABAAR channel blocker PTX (100 μM) (Supplementary Fig. 4a). This drug caused a similar shift in holding current in ​Pin1−/− and ​Pin1+/+ mice (Supplementary Fig. 4b,c), indicating that extrasynaptic GABAA receptors are not influenced by ​Pin1-mediated signalling.


The present study shows that ​NL2 is a newly identified substrate of proline-directed phosphorylation. This post-translational modification, acting on its unique ​Pin1 consensus motif localized within the CD (S714-P), modulates the amount of ​NL2–​gephyrin complexes at synaptic sites. This modulation impacts on GABAergic transmission, by selectively affecting the total number of synaptic GABAARs. On the basis of these findings, post-phosphorylation prolyl-isomerization can play a crucial role in remodelling the GABAergic PSD to sustain plasticity processes.

Protein phosphorylation on ​serine and ​threonine residues preceding a ​proline, the so-called proline-directed phosphorylation, has emerged as a mechanism regulating signalling events through conformational changes that are catalysed by the phospho-dependent recruitment of the peptidyl-prolyl isomerase ​Pin1. While the different roles of ​Pin1 in dividing cells have long been established and characterized19, its function in post-mitotic neurons in general and at synapses in particular is still poorly understood. In a previous study, we identified ​gephyrin, the main scaffolding protein of inhibitory PSD, as a new target of post-phosphorylation prolyl-isomerization22.

Here, by inspecting the protein sequence of NL CDs, we identified S/T-P motifs that may provide ​Pin1 binding sites if phosphorylated in vivo. In particular, ​NL2 presents a unique ​Pin1 consensus site in its cytoplasmic region, S714-P, which is located 15 amino acids apart from the transmembrane domain. Even though this proximity to the plasma membrane raises doubts about its accessibility by a proline-directed kinase, several lines of evidence suggest that endogenous ​NL2 can undergo proline-directed phosphorylation. First, this isoform was recognized by the MPM2 antibody on ​NL2 immunoprecipitation from mouse brain homogenates. Second, MPM2-mediated ​NL2 immunoprecipitation was still maintained on removal of the ​NL2–gephyrin-binding domain, excluding the possibility of an indirect recognition mediated by endogenous ​gephyrin. Third, such detection was completely lost on ​NL2HA-S714A mutagenesis. This phosphorylation event is then able to directly recruit the effector molecule of the signalling cascade ​Pin1, as shown by co-immunoprecipitation experiments with endogenous neuronal proteins. Also in this case, ​Pin1 binding to ​NL2 was still maintained on the removal of the GBD, while it was completely abolished by mutating S714 to ​alanine, thus suggesting that the prolyl isomerase can be directly recruited by the unique ​NL2Pin1 consensus motif in a phosphorylation-dependent manner. These results altogether indicate that ​NL2 represents a newly identified substrate for proline-directed signalling cascade in vivo.

Our biochemical data demonstrate that ​NL2–​gephyrin interaction is negatively regulated by proline-directed phosphorylation. Co-immunoprecipitation experiments on recombinantly expressed ​gephyrin-​FLAG and ​NL2HA unveiled an enhanced complex formation on pharmacological inhibition of ​Pin1 catalytic activity. Similarly, endogenous ​NL2/​gephyrin complexes pulled down from whole brain or hippocampal tissues of ​Pin1−/− animals were significantly augmented as compared with the corresponding WT tissues. These biochemical findings were also validated by immunocytochemistry performed on cultured hippocampal neurons, where we could detect a high number of clusters co-labelled for ​NL2 and gephryin as well as their increased apposition to presynaptic GABAergic inputs in the absence of ​Pin1 expression. Interestingly, the ​NL2 point mutant unable to undergo prolyl-isomerization was capable to recruit ​gephyrin even more efficiently as compared with the WT form, whereas ​gephyrin mutagenesis at two putative ​Pin1 consensus motifs, S270A and S319A, located within, or close to, the minimal ​NL2 binding domain, was completely ineffective. The fact that this post-translational modification seems to control the strength of ​NL2 association with ​gephyrin by acting mainly on ​NL2, and not vice versa, further reinforces the emerging idea that cell adhesion molecules are key determinant in regulating synapse function. In a recent study by Giannone et al.18, it has been demonstrated that the level of ​NL1 phosphorylation at a specific ​tyrosine residue located within the GBD dictates the strength of ​NL1/​gephyrin interaction. In other words, ​NL1, the isoform enriched at excitatory synapses and therefore mostly associated with ​PSD95, can potentially recruit ​gephyrin as well as ​NL2, but its phosphorylation, promoted by neurexin–adhesion signalling, precludes such interaction while favouring ​PSD95 binding. Our experimental data indicate that proline-directed phosphorylation is acting similarly to ​tyrosine phosphorylation signalling. Since ​NL2 S714 is not positioned within the GBD, but is located just 50 amino acid upstream, it is reasonable to believe that ​Pin1-driven conformational changes, by affecting the overall folding of the CD, will induce ​gephyrin release (Fig. 10a). Alternatively, these conformational changes may promote ​NL2tyrosine phosphorylation, an event shown to impede NLs/​gephyrin interaction18 (Fig. 10b). Interestingly, ​tyrosine to ​alanine mutagenesis on ​NL2 was shown to completely abolish recombinant ​gephyrin recruitment by the mutant protein or to strongly reduce its interaction with endogenous ​gephyrin6. Whether ​NL2 phosphorylation occurs at tyrosine 770 and whether this event is able to hamper ​gephyrin binding is still unknown.
Figure 10: Model of the putative cross-talk between proline-directed phosphorylation and ​tyrosine phosphorylation.
Model of the putative cross-talk between proline-directed phosphorylation and tyrosine phosphorylation.
Phosphorylation of ​NL2 CD at S714 by a proline-directed kinase allows the recruitment of the proly isomerase ​Pin1. ​Pin1-driven conformational changes, by altering the folding of the ​NL2 CD, may represent the main cause responsible for ​gephyrin detachment (a). Alternatively, ​Pin1-mediated structural rearrangement may render the conserved ​tyrosine residue of the GBD (Y770) susceptible to phosphorylation, an event shown to prevent ​NL1/​gephyrin interaction (b).
The other partner of the complex is represented by ​gephyrin, a recognized target of ​Pin1 (ref. 22). ​Gephyrin contains 10 consensus motifs mostly concentrated in its C-domain, and all of them found to be phosphorylated in vivo35, 36. This region of the protein is positioned between the amino-terminal G- and carboxyl-terminal E-domains, which are directly involved in ​gephyrin multimerization. Conformational changes induced by phosphorylation, possibly followed by prolyl-isomerization, are expected to alter the conformation of the ​gephyrin C-domain and in turn, regulate specific functional properties of ​gephyrin, in particular its binding to interacting proteins, including possibly ​NL2. However, the complexity of the system under investigation makes it very difficult to determine whether and how a specific phosphorylation event can contribute, directly or indirectly, to enhance ​gephyrin association to ​NL2. Nevertheless, it should be emphasized that ​gephyrin is robustly phosphorylated at several residues in vivo, thus suggesting that a specific pattern of phosphorylation, rather than a single post-translational modification, is functionally determinant. In contrast, ​NL2 possesses a unique target for prolyl-isomerization suggesting that it could represent the master switch of the signalling cascade.

Our electrophysiological experiments clearly demonstrate that deletion of ​Pin1 specifically affects GABAergic transmission, causing a dramatic increase in amplitude, but not in frequency, of sIPSCs due to an increase in the number of GABAARs at post-synaptic sites. Notably, such enhancement was detected on neuronal overexpression of the ​NL2 mutant unable to undergo prolyl-isomerization, suggesting a functional link between the signalling cascade strengthening ​NL2/​gephyrin interaction and the increased synaptic recruitment of GABAARs. There is a large body of evidence underlying the key role played by ​NL2 in promoting clustering and/or stabilization of GABAARs at post-synaptic sites. By employing a heterologous expression system, it was shown that GABAARs are able to co-aggregate with ​NL2 and only the presence of this isoform can induce strong GABAergic presynaptic differentiation from co-cultured neurons and promote the establishment of fully functional hemi-synapses37. In ​NL2-deficient mice, the number of functional GABAARs detected in the retina was shown to be drastically reduced38. Furthermore, targeting of GABAARs and ​gephyrin scaffold appeared severely compromised in the pyramidal cell layer of the CA1 region of the hippocampus, a morphological phenotype accompanied by a strong deficit in synaptic inhibition6.

The increased recruitment of synaptic GABAA receptors in ​Pin1−/− mice may simply depend on the enhanced ​gephyrin targeting at synaptic sites. More scaffold deposition should offer a high number of binding sites available for the transient immobilization of GABAARs at inhibitory synapses. In addition, or alternatively, we cannot exclude the possibility that the extracellular domain of ​NL2 could also participate in GABAARs receptor recruitment. The unique S714-P consensus motif, located very close to the ​NL2 transmembrane domain, could influence the folding of the extracellular domain of ​NL2, rendering it incapable to interact in cis with GABAAR subunits. This type of mechanism has been shown to operate at excitatory synapses, where the abundance of NMDARs is controlled by the interaction occurring between the GluN1 subunit with ​NL1-specific sequences located in its extracellular domain39.

In conclusion, our findings unveil the existence of a new signalling pathway operating at GABAergic synapses to alter the efficacy of GABAergic transmission by modulating ​NL2/​gephyrin interaction. Although a comprehensive understanding of the molecular mechanisms underlying the action of ​Pin1 on ​NL2/​gephyrin interaction is still lacking, we believe that our study further emphasizes the key role played by ​NL2 in organizing and stabilizing GABAergic synapses.

How to cite this article: Antonelli, R. et al. ​Pin1-dependent signalling negatively affects GABAergic transmission by modulating ​neuroligin2/​gephyrin interaction. Nat. Commun. 5:5066 doi: 10.1038/ncomms6066 (2014).