– From twin studies to discoveries of dendrites protein production
As told by Erin Schuman

Formative years in SoCal
I grew up in Huntington Beach which, at the time, was a rather sleepy surfer’s haven in Southern California. I attended local Catholic Schools.My mother, Susan Arant, a school teacher for over 35 years, was my 8th grade teacher and she was an enthusiastic supporter of my schooling and early intellectual life. She divorced my biological father, Bruce Chandler, when I was very young and I sadly had little contact with him, but have recently been enjoying getting to know members of the extended family.

For my senior honors thesis, I designed my own study to measure memory abilities in identical and fraternal twin kids, driving my VW bug around Southern California to test young kids in their homes, a rather chaotic experience

A grandfather's influence
My grandfather, an engineer who designed some of the pumps for the Hoover Dam on the Colorado River, was my earliest tutor. We worked on math and physics problems together – I still remember his elegant fingers scratching equations on a pad and the science articles he would cut out from the L.A. times for me to read. I was an undergraduate at the University of Southern California and originally enrolled in a pre-medical course, but with an interest in the mind and how brains work, I navigated to the Psychology department. I was lucky to have as a mentor a new Assistant Professor, Dr. Laura Baker, a behavioral geneticist who studied cognitive abilities in human twins. As my time at USC progressed, I became more and more fascinated with the brain- and memory formation. For my senior honors thesis, I designed my own study to measure memory abilities in 7-12 year-old identical and fraternal twins, recruiting subjects locally and driving my VW bug around Southern California to test young kids in their homes, a rather chaotic experience.

Two things learned
After writing my USC undergraduate honors thesis, I was convinced of two things: that I wanted to do research on the brain substrates of learning and memory and that humans were not my preferred experimental subjects. At that time, simple forms of associative learning had been demonstrated in a few marine invertebrate species, and, and their brains could even be studied directly with electrophysiological recordings. For my PhD, I joined a lab at Princeton University and began to study associative learning in Hermissenda crassicornis, teaching these marine snails to move less vigorously towards light after it had been paired with turbulence. I became an electrophysiologist and discovered that the enduring nature of a Hermissenda memory was due to the persistent activity of a protein kinase, acting on two different brain potassium channels.

Midway through my PhD studies, my advisor was denied tenure and moved the lab (just me and him at that point) to the Midwest. I loved recording from neurons but my relationship with my supervisor was very dispiriting and isolating. I thought about quitting science. Luckily, in the summer that my PhD supervisor moved to Indiana University, I took the Neural Systems and Behavior course at the legendary Marine Biological Labs in Woods Hole, Massachusetts.

Working as a post-doc in the Madison lab, I felt like I was on a scientific joy-ride in comparison to my dismal graduate school experience

The course directors were Tom Carew and Darcy Kelly and there was an incredible line-up of faculty, people like Russ Fernald, Ron Calabrese, Jack Byrne and Eduardo Macagno, who all re-affirmed my love of science. They must have recognized something promising in me and they encouraged me to continue. Later, Tom Carew was instrumental in my moving back to Princeton to write up my PhD thesis and helped me find a great postdoctoral environment – working with Dan Madison at Stanford.

Working as a post-doc in the Madison lab, I felt like I was on a scientific joy-ride in comparison to my dismal graduate school experience. The Molecular and Cellular Physiology department at Stanford was newly formed and there were lots of smart, interesting and colleagues who became life-long friends. I joined Dan’s lab because I was still interested in plasticity and keen to discover mechanisms using electrophysiological recordings in hippocampal brain slices. The concept of a retrograde messenger had been invoked in the long-term potentiation (LTP) field to coordinate plasticity that was induced in the postsynaptic cell with eventual presynaptic changes. I showed that the gas nitric oxide (NO) could serve as such a retrograde messenger during LTP in hippocampal brain slices. Jane Haley, Paul Chapman, Tom O’Dell and Eric Kandel published similar data around the same time. In a follow-up study, that involved paired intracellular recordings from neighboring neurons, I found that LTP induced in one cell could spread to nearby, but not distant, synapses, consistent with the actions of a diffusible messenger.

I remember those days at Stanford as some of the best times in my career – I discovered how much I love doing experiments – and how exhilarating science can be in the right environment

Dan was an extremely supportive and smart boss and he was a great teacher too. Sadly, he passed away recently, way too young, and will be missed by so many scientists and friends. I remember those days at Stanford as some of the best times in my career – I discovered how much I love doing experiments – and how exhilarating science can be in the right environment.

After our first NO paper came out, Dan went to give a seminar at Caltech and he came back and shared that some Caltech colleagues had suggested that I apply for an Assistant Professor position in the Division of Biology that had been open for some time. Applying for professorships was very far from my mind – I was having so much fun doing experiments and thought of doing a second postdoc. I applied and then interviewed for the Caltech job – the experience was not pleasant. During my job talk, two senior faculty members were shaking their heads, whispering, and interjecting negative comments outloud. (Later, I learned that a former PhD student of one of those faculty was also a candidate for the position). After that, I went back to my hotel and told Dan “Even if they offer it to me, I will never come here”.

I bought a used VW convertible and I have potent memories of driving home late from the lab on paradoxically warm Pasadena winter nights and inhaling the intense smells of citrus blooming

I was thus surprised when the job was offered to me and after some kind exchanges with other faculty members, I was convinced to join the Caltech faculty. I opened my lab in the Beckman Behavioral Biology building in 1993. At the beginning, I really missed the camaraderie of my Stanford buddies doing experiments together. I dealt with the change by immersing myself in the lab set-up and getting experiments going. I bought a used VW convertible and I have potent memories of driving home late from the lab on paradoxically warm Pasadena winter nights and inhaling the intense smells of citrus blooming.

Summercourtship began with building lab rig
It was during these early years that I met my brilliant life-long partner and husband, Gilles Laurent, a systems Neuroscientist, who was already on the Caltech faculty. Our courtship began when Gilles asked if he could build an electrophysiological rig in my lab over the summer. Amongst the many friends and colleagues I eventually made, I found a fabulous mentor in the late Norman Davidson, a chemist by training, who turned to neurobiology later in life.

Our first discovery
I was very lucky with my first graduate student, Hyejin Kang. She had done lab rotations in a few labs before I arrived on campus, but none of these “clicked”.We hit it off straight away and started doing experiments together. She was super smart and very hard-working. She was also resourceful – she canvassed the lab and took over any electrophysiology set-up that wasn’t in use.I remember hearing her run between rooms to change solutions at just the right time on each experiment.

Together, we set out to explore whether some of the same molecules that sculpt neurons and their connections during development might also participate in changing synapses in adult animals. We applied a growth factor (brain-derived neurotrophic factor; BDNF) to brain slices and found that it caused a rapid and long-lasting enhancement of synaptic transmission. We knew from other behavioral and synaptic plasticity studies that long-term memories require newly synthesized proteins. So we did a simple experiment- we added a chemical inhibitor to block protein synthesis together with BDNF.We got a surprising result. The enhancement of synaptic strength by BDNF was immediately blocked – not just the long-term plasticity. The synapses we were recording from were a few hundred microns away from the cell body, too far away to allow for the transport of the new proteins so quickly – suggesting that the protein synthesis source was not the cell body, but local, near the synapses. We went on to show this directly by isolating the synapses from the cell bodies and doing the experiment again.

Oswald Steward and colleagues had detected poly-ribosomes, but most neuroscientists still believed that all proteins were made in neuronal cell bodies and then transported to synapses.

In this “soma-free” slice preparation, the plasticity could still be elicited and still showed the early requirement for protein synthesis. The conclusion that proteins made near synapses are required for plasticity surprised nearly everyone. More than a decade earlier, Os Steward and colleagues had detected poly-ribosomes (multiple ribosomes translating a single mRNA) near synapses, but most neuroscientists still believed that all proteins were made in neuronal cell bodies and then transported to synapses. Indeed, when I first shared the data the idea was called “crazy” by more than one of my colleagues.

Backed up by Marting and Holt
Luckily, around the same time, Kelsey Martin was working in Eric Kandel’s lab and she found that local protein synthesis was required for plasticity at the sensory to motoneuron synapse in Aplysia. Meanwhile, Christine Holt was working on the mechanisms that allow axonal growth cones to turn towards their targets- she found that local translation and degradation of proteins was essential for the appropriate turning decision.

When I first shared the data the idea was called “crazy” by more than one of my colleagues

Seeing is believing
At that time, I recognized the importance of visualizing protein synthesis directly in the dendrites. I felt with conviction that if we could see new proteins emerge in the dendrites we would really convince ourselves and others that proteins can be made locally. I had the idea of creating a GFP-based reporter- where the mRNA could be targeted to the dendrites. We would then visualize GFP fluorescence pop up in the dendrites- providing proof for local synthesis.

I remember sharing this idea with a senior colleague at Caltech and she said, “That will never work!”.

The GFP reporter in fact worked beautifully and we coupled it with delicate “neurosurgery” – delivering a small cut to isolate the dendrites from the cell body. With this, we provided direct evidence for local translation. Around that time, Daniela Dieterich joined my lab as a postdoctoral fellow. She forged a collaboration with my colleague Dave Tirrell in Caltech’s chemistry department.

Dave had been developing non-canonical amino acids, mostly methionine derivatives (e.g. azidohomoalanine, AHA), which were functionalized with azide or alkyne groups and could be charged onto methionyl tRNAs by the cellular methionyl tRNA synthetase. Dani showed first in living cells treated with AHA that newly synthesized (AHA-bearing) proteins could be isolated and purified using click chemistry and then identified using mass spectrometry. We called this method biorthogonal non-canonical amino acid tagging (BONCAT) and it has been invaluable to us and many others. Using azide or alkyne-bearing fluorescent tags, Dani then showed that nascent proteins could also be directly visualized (FUNCAT). Later, with Dave, Dani, Cyril Hanus and Beatriz Alvarez-Castelao, we modified the tRNA synthetase and its cognate amino acid to make the system work in a cell type-specific manner and now there are platforms available in mouse, fly, worm and zebrafish.

Sabbatical year in Paris
In 2008, Gilles and I took a sabbatical in Paris. I worked in Antoine Triller’s lab at the Ecole Normale Superieure. There I learned single particle molecular tracking and used FUNCAT to visualize the movements of newly synthesized proteins within living synapses. We could see, for the first time, nascent labelled proteins moving in the plasma membrane.

During our time in Paris we were approached by colleagues from the Max Planck Society to gauge our interest in building and directing a new Max Planck Institute for Brain Research in Frankfurt. In 2009, after ~35 years of joint experience on the faculty at Caltech, with differing levels of discontent about doing science in the U.S. and with a big spirit of adventure, Gilles and I decided to move to Germany with two of our daughters.

Together with the architects and building team we built a beautiful space that is now a high-energy, interactive neuroscience institute.

mRNA localization more of a rule than an exception
The first experiment we did after the lab moved to Frankfurt was to use emerging next generation sequencing technology to directly sequence the mRNAs present in the neuropil – this was spearheaded by Ivan Cajigas and Georgi Tushev. At that time, the potential impact of local translation was severely hampered by the very limited number (~25) of mRNAs that had been detected in dendrites. The laminar nature of the hippocampus was perfect for the isolation of the neuropil (enriched in axons and dendrites) and the somata from brain slices.Together we microdissected hundreds of slices and discovered that the mRNA population was not in the 10s or even 100s, but rather in the thousands. Our first RNA-seq experiments identified ~2500 mRNAs- and as the technology improved this number climbed to ~ 5000 species. There was convergence on this big number of localized mRNAs from all of the groups who were sequencing axonal and dendritic transcripts at the time, including Kelsey Martin’s and Christine Holt’s lab. This huge number surprised us – and strongly suggested that local translation is used for the constitutive function of synapses, not just invoked during special cases of plasticity. Anne Biever and Caspar Glock’s later work describing the local translatome (all mRNAs in the process of active translation) confirmed this – all the mRNAs detected were also translated under basal conditions. In addition, by comparing the magnitude of the somatic and local translatomes, we learned that the neuropil is the primary site of synthesis for over 800 synaptic proteins.

But where are the machines?
Confronted with such an abundance of protein synthesis we had to ask ourselves about the machines (ribosomes) that make the proteins.As I mentioned earlier, Os Steward had detected polyribosomes near synapses in the 1980’s and then Kristen Harris with Linnea Ostroff, Jenn Bourne and others had conducted beautiful detailed studies of the abundance of ribosomes in dendrites using serial-section electron microscopy. Although they could see that polyribosomes were more abundant at synapses after plasticity, there were simply too few ribosomes detected to account for all of the locally synthesized proteins we could see.We reasoned there might be a ”hidden” population of individual ribosomes present at synapses- that were not detectable in the standard EM experiments.Using super-resolution imaging, biochemical experiments, and most recently, cryo-electron tomography (cryo-ET) we indeed detected many more ribosomes, including individual ribosomes or “monosomes”.With the amazing resolution of cryoET, not only can we see individual ribosomes within the neuron, we can also resolve whether they are actively translating a protein or not.Together with Andre Schwarz and Mara Mueller, we are beginning to see important differences in the “local” ribosomes that may render them sensitive to neuronal activity.In the future, I’m very keen on using cryoET to visualize and understand protein synthesis and other cellular processes in their native neuronal context.

Alongside the “basic” work we have done on how synapses get the proteins they need to function and adapt has been an increasing appreciation that many neurological disorders, including neurodevelopment and neurodegenerative diseases, manifest themselves in dysfunction at the synapse, or “synaptopathies”.In the coming years, I hope that many of the basic discoveries that we and others have made about how synapses work will increasingly be applied to reveal what goes wrong in neurons and at synapses in disease.I’m thrilled to become an active contributor to this work as I will soon join University College London as the Director of the Institute of Neurology (IoN) - an Institute with brilliant and vibrant colleagues who truly move the basic discoveries of the lab into clinical applications that improve human lives- all in one place.

It is an enormous privilege to be a scientist

It is an enormous privilege to be a scientist. One of the greatest pleasures has been to work alongside and discover new things with the many, very clever, driven, interesting and fun people that made my lab what it is. Without them, none of the work would have been possible.

In addition, I have to also acknowledge how important it has been to share this life journey with my fabulous partner, Gilles Laurent, and our wonderfully talented, smart, sensitive and very funny daughters, Emma, Charlotte and Camille, who are also currently pursuing careers in science.

(Some text of this autobiography has previously appeared in Erin Schuman’s biography for the Lundbeck Brain Prize and the Nakasone Prize Science supplement).