Feature: Wired to think

Since the time of the great Spanish neuroanatomist Ramon y Cajal, nearly 130 years ago, we’ve known that the brain is an amazingly complex organ, in much the same hand-waving way that we know a blue whale is impressively large.

Australian neuroscientist Professor Seth Grant, of the University of Edinburgh, says it has become clear in the past decade that the human brain, and all animal brains, are vastly more complex than we thought, or could have even believed

Grant, who is a plenary speaker at ComBio 2012 in Adelaide, was a pioneer in a new field of brain science in the late 1980s: the science of the synapse. Two decades on, he says, it is clear that the synapse is an extremely complex entity in its own right.

According to Grant, the completion of the Human Genome Project in 2003 and the advent of new high-throughput proteomics tools has lent new impetus to the field of synaptic proteomics.

He believes the workings of the brain and the large number of brain pathologies in humans will eventually be understood in terms of the interplay of highly organised networks of synaptic proteins, especially within the unsuspected diversity in proteins that form the post-synaptic density – the generic term for post-synaptic proteins (PSPs).

Professor Seth Grant

He and his colleagues have traced the evolution of the synaptic proteome from the time of the first complex multicellular organisms, Cnidarians (jellyfish) and Ctenophorans (comb jellies), 580 million years ago and shown a step-wise increase in the complexity of synapse proteins as new and more complex life forms emerged, down to relatively recent times and the divergence of humans from our great ape relatives.

An adult human brain comprises some 100 billion (1011) neurons, each intricately networked with around 10,000 others, via fine, thread-like dendrites. In total, the dendrites carry around 100 trillion (1014) synapses.

As a young molecular biologist at New York’s Columbia University in 1992, Grant was lead author on a paper in Science which reported that a transgenic knockout mouse lacking a functional gene for a synapse-associated protein called fyn, normally expressed in the hippocampus, the brain’s memory-forming centre, exhibited impaired learning and a spatial memory deficit.

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Fyn was first in what became a fast-growing catalogue of synapse-associated proteins. The Science paper, along with another by a MIT research team, “started things off” says Grant. Today, the number of kown postsynaptic proteins in the human brain runs to more than 1000 and the number is still increasing.

“When I started working in the field in the late 1980s, virtually nothing was known about the molecules in the synapse,” says Grant. “It seemed an interesting new area, where we could bring all kinds of new molecular tools and methods to bear on unresolved questions.”

Fundamental units

“During the 1980s and earlier, synapses were recognised as fundamental units of the nervous system that operated via neurotransmitters and were targets for brain-acting drugs. But the genes encoding synaptic receptors, and other synaptic proteins, were almost unknown in the late 1980s.

“I went from the University of Sydney to Cold Spring Harbour in 1985 and worked on developing transgenic mice. I decided to apply what I had learned to neuroscience, and left for Columbia University in 1989 to work as a post-doc in Eric Kandel’s lab – he won a Nobel Prize in 2000.

Schematic model of the structure of a vertebrate proto-synapse. The components aggregate to form a macromolecular structure tethered to the cell membrane. This transduces environmental information via receptors and transmits it to intracellular biological processes and pathways, including to the nucleus, where it modulates gene activity in response to the stimulus.

“Over the next few years we demonstrated the involvement of some of the protein molecules in brain plasticity, memory and learning, and published the fyn paper in 1992. Since then, hundreds of papers using the same approaches have been published.

“I then took up a post at Edinburgh University in 1994 and was the first to apply proteomic methods to studying the synapse. Proteomics was very important: we could understand, for the first time, what synapses are made from and how neurotransmitters worked at whole new level of detail.”

In 2000, Grant’s Edinburgh University team, working with several researchers from Glaxo-Wellcome R&D reported in Nature Neuroscience that they had isolated an N-Methyl-D-Aspartate receptor (NMDAR) multi-protein complex from mouse brain, one of receptors for the excitatory neurotransmitter glutamate.

The complex consisted of 77 proteins organised into a receptor, adaptor, signalling, cytoskeletal and several other novel proteins, revealing that neurotransmitter receptors were actually just a part of much bigger molecular machines made of many other proteins.

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“The complex comprised about 10 times as many proteins as we expected,” he says. “It shocked us, and everybody else. We subsequently expanded on this work, studying synapses in multiple species with proteomics, including in humans, where there are over 1000 proteins in the post-synaptic proteome.”

Grant says the broad biological explanation for the complexity of PSPs is that all cells need to monitor and respond to changes in their environment, and have evolved molecular machinery for processing information and conveying it from the receptors on the surface through biochemical pathways into the nucleus and genome.

“What is most striking about the post-synaptic proteome is that the majority of key components of synapses actually evolved prior to the evolution of multicellular organisms, in single-celled eukaryotic organisms. In other words, the key molecular machinery in synapses actually evolved before the origins of nerve cells and brains.

“When you’re studying proteins involved in higher cognition, learning and memory in humans, you have an expectation that they will be magnificent molecules, that special proteins conferred specially capacities on the human brain. But that turns out not to be the case.

“It’s not surprising that most people thought the human brain would be very special, but its commonality with the brains of other vertebrates is quite extraordinary. People who expected humans would be specially equipped are sometimes a bit disappointed, even though these findings illuminate common molecular machinery across all animals.

“One of biology’s great correlations links brain size and intelligence. The dominant theory has been that as the nervous systems acquires more neurons, brain function increases. It appeals to our logic, but modern humans actually have smaller brains than Neanderthals did. We should pay particular attention to scientific observations that dissociate or break correlations, because they are worth hundreds of other correlations.”

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From genes to behaviour

According to Grant, apart from the insights it has provided into brain evolution, a detailed knowledge of the molecular composition of synapses provides a “fabulous platform” for exploring the role of synaptic protein mutations to brain disorders such as schizophrenia and bipolar disorders.

“Again, it’s plausible that synapses will be found to be important. We know that all sorts of drugs act on synapses, but we don’t know what role mutations play in the aetiology of brain disorders. One way to explore that, using our new knowledge of the proteome of the synapse, is to ask what is known about Mendelian genetic disorders and their impact on the synapse.

“As the number of synapse proteins has increased, we have identified several hundred genes for PSP proteins that are mutated in some way, and shown they are associated with over 130 different brain disorders. That opens a Pandora’s box of disease biology. It reinforces evidence that there is something very special about the way synapses work. That’s extremely important, because there are practical things we can do with such information.

“We need that many genes because all those molecules are doing things that subtly impact and control the way synapses work. When these complex systems go wrong, they cause brain disorders. I’m particularly interested in understanding what the synapse is doing in the context of behaviour. One form of behaviour – instinctive behaviour – has been neglected over the past 50 years.

“People like Niko Tinbergen, Konrad Lorenz and Karl von Frisch studied innate behaviour – instinct – yet we still know almost nothing about the neural basis of instinctive responses that animals deploy, in a stereotypical way, when they move from one environment to another.

“For example, when a mouse pops its head out of a hole, it typically comes only part way out and has a look around, to check if something bad might happen. It doesn’t do so as a result of some logical process, it’s an instinctive response.

“Place a cat near a mouse, and the mouse will either make a run for it, or freeze. These behaviours are instinctive, initiated by some sort of sensory input. The brain generates an instinctive response to the stimuli, but nobody has asked how the response is encoded and how it is elicited by the stimulus.”

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According to Grant, the second major area of behavioural biology that has yet to be explored is the nature of our behavioural repertoire. Over its lifetime, every animal displays a wide range of behavioural responses in different situations, some innate and other learned. “If we understand the organisation of the post-synaptic proteome, we should be able to understand the correlates of different behaviours at the molecular level.”

Asked if it would eventually be possible to predict an individual’s behavioural responses from combinations of post-synaptic proteins, Grant says: “I believe we’re on the cusp of doing that already. We have found evidence for these combinatorial properties. For example, one of the key concepts that comes out of that is that different forms of glutamate signalling recruit particular combinations of synaptic proteins, which induce signalling via different pathways.”

One of the exciting things that could flow from understanding the molecular basis of behaviour at this level is that drugs that target particular proteins could have beneficial therapeutic effects across multiple brain disorders.

“I think the days are numbered for the traditional approach of diagnosing psychiatric disorders from standard symptoms. Our thinking needs to be more genetically oriented. Every single disease is likely to be made up of multiple disease causing gene mutations that affect cognition, motor function, and memory.”