Shortly after the burst

Prof. Mike Greenberg talks about his research on Immediate Early Genes

Despite similarities in the numbers of genes and structure of neural circuits, primates have evolved vastly more complex brains and behaviors. What do those differences look like in the brain? A recent paper from the labs of Michael Greenberg and Margaret Livingstone at Harvard Medical School examines how a short (85 base pairs!) sequence in the regulatory region of the OSTN gene, which was previously known as a secreted protein in bone and muscle development, has allowed it to be expressed in the brains of primates, but not those of rodents. The expression of OSTN is special for another reason: it is one of the first primate-specific genes regulated by immediate early genes (IEGs) to be found. IEGs are a group of genes whose transcription in neurons is transient and commonly follows a burst of spiking activity. In their short window of expression many IEGs are known to regulate expression of specific downstream genes. The new paper from the Greenberg and Livingstone labs gives us a peak into how small differences in common molecular pathways may be implicated in the diversity of species. The journey of our knowledge and understanding of IEGs and the genetic response to neural activity is also the journey of the scientist who first observed the expression patterns of IEGs, and who has since dedicated a great deal of his scientific career to investigating them: Professor Michael Greenberg. A few weeks ago, I had the pleasure of sitting down with him to talk about his work, past, present and future.

golgistainedpyramidalcell

Golgi stain of human pyramidal neuron

Michael Greenberg started his career as a molecular biologist, interested in how cells that have stopped replicating get stimulated to re-enter the cell cycle. To this end, during his post-doc in the lab of Ed Ziff at Rockefeller University, he studied how proto-oncogenes are involved in this mechanism. In 1984, he made the unprecedented finding that administration of growth factors to cultured fibroblasts induced a quick rise in the transcription of c-fos gene that lasted for about 30 minutes. Both the speed with which transcription is initiated after stimulation, and with which it is terminated are unusual. For comparison, some other mRNAs are either transcribed at constant rates or in periods that last for hours. Soon thereafter, he discovered that the peculiar expression pattern of c-fos in neurons could also be induced by activation of L-type voltage-dependent calcium channels. The interest in his findings boomed in those years, and he found himself having to navigate the field to find the niche in which to start his own lab. When talking about those years, he said: “like many postdoctoral fellows making the transition to faculty I had to think very hard about how I was going to differentiate myself from other scientists at my institution and around the world and it became clear that despite the fact that I had made this discovery of c-fos induction and I that I had since thought of it as my discovery there were many other researchers who wanted to follow up on it in the context of cell cycle progression. In an effort to distinguish myself, I changed directions to the question of how activity through fos regulates transcription and articulated a vision as to how that might play a role in nervous system development and function.”

The first question he addressed in his lab was trying to understand how growth factors (specifically BDNF in the brain) and L-type channels induced the transcriptional surge in c-fos. In this time, his work flowed in parallel with many researchers who were investigating the fos-activating molecular pathways in other cell types. “It was feasible because the methodologies for studying and addressing that question were available and initially what it really amounted to was taking the promoter and mutating it and asking what are the elements within the fos promotor that are important for activity or for a neurotrophic factor response.” It was the late ‘80s and this approach to understanding IEGs was facilitated by the advances in techniques such as mutagenesis, reporter constructs, gel mobility shift assays, and immune-labelling using antibodies. These allowed scientists to detect factors that bind to critical regulatory elements, to purify them, to visualize proteins in tissue, “to get into the initial mechanism by which neuronal activity through calcium turned on the fos gene and then other genes as well. The function of fos was much more difficult to get at because it was a complex family and what it does in a cell depends on the cell-type it is activated in.” They characterized the partnership of members of the fos family with jun, another group of IEGs, and how they acted together in the transcriptional regulation of their downstream targets. It was apparent that the surge of IEGs was necessary to link the expression of their downstream targets to neuronal activity, but what these targets were remained a mystery.

In the years to follow, fos and other IEGs became famous as a tool in neuroscience because of their peculiar properties. One could use them to specifically label the cells that were most recently activated. The questions as to what fos is doing in a cell, and how it may play different roles in different components of the circuit became dormant. In recent years Michael Greenberg and many other researchers have been revisiting these topics. New advances in how we can manipulate the genome now allow us to label subsets of neurons; to insert or delete genes at precise time points or in specific groups of cells; or to initiate or silence spiking in neurons. Our access to biological systems and our ability to manipulate them is fueling our curiosity. Not only can we now ask what fos is doing in a cell, but also what it’s doing in a circuit and how it affects the relationship between two neurons. The latest research on the activity of IEGs has added a further layer of complexity and understanding to plasticity, circuit homeostasis, and the short and long term consequences of neural activity.

Michael Greenberg gave me some insight into how he thinks these processes act and what is to be investigated next. One of the key findings of the recent years is that IEGs “will be turned on in pretty much every cell type in response to many different stimuli. Somehow they need to gain specificity, and the way they get specificity is by choosing different enhancer sequences in each type of cell and maybe different enhancer sequences in response to different stimuli”. An example of this is the regulation through IEGs of the expression of different growth factors in excitatory and inhibitory neurons in response to neuronal stimulation. When a cell experiences a surge in excitation, and fires at a high rate it runs the risk of overexciting, a process known as excitotoxicity. IEGs provide one mechanism that cells use to mitigate excitation. In excitatory neurons, IEGs regulate BDNF, which the cell then uses as “a beacon for the recruitment of inhibitory synapses onto the excitatory neuron.” In one class of inhibitory neurons (known as VIP) it regulates IGF1, which also promotes inhibition onto the cell itself. The VIP neuron, however, is special because it synapses onto the somatostatin inhibitory cells that then synapse onto the excitatory cells. Following expression of IGF1 the VIP neuron decreases its output, thereby disinhibiting the somatostatin neuron and allowing it to more strongly inhibit the excitatory cell. Both these mechanisms have a common effect: an increase in inhibition on the excitatory cell! This is an example of how IEGs might mediate circuit homeostasis.

Fos is implicated in other activity-dependent transcriptional pathway as well. In response to synaptic activity, neurons have been found to employ local translation of mRNA into proteins that can be rapidly recruited to the synaptic membrane. This allows for very precise and targeted regulation of protein expression at active synapses. An example of this is ARC, which is locally translated and “promotes the internalization of the AMPA subtype of glutamate receptors in response to activity”. This elegant mechanism requires a “restocking of the shelves”, a process by which the local mRNA is replenished, in part also because local mRNA tends to be more readily degraded than mRNA in the soma. Could fos or other IEGs regulate after a burst of spiking activity the transcription of more ARC mRNA  to be shuttled back to the postsynaptic site?

Our growing understanding of IEGs shows that they play important roles in neuronal circuit dynamics and homeostasis, roles that may have implications in a vast range of behaviors and cognitive processes. IEGs are a complex family of transcriptional regulators, with differential expression patterns and targets in different cells; this characteristic allows for this common strategy to adapt to needs in each part of the circuit, and to contribute to the diversity of neural cell types. Michael Greenberg’s latest paper suggests that the diversity in the targets of this quick transcriptional response to cellular activity may be implicated in evolutionary differences among species as well. “We want to know what the factors are in human neurons because our brain is much more complex and is endowed with the properties that are interesting and special to the human or the primate; now we’re trying to figure out how evolution has selected this network and works with it to endow us with human primate activity dependent responses.”

Bibliography

  1. Ataman B, Boutling GL, Harmin DA, Yang MG, Baker-Salisboury M, Yap E, Malik AN, Mei K, Rubin AA, Spiegel I, Durresi E, Sharma N, Hu LS, Pletikos M, Griffith EC, Partlow JN, Stevens CR, Adli M, Chahrour M, Sestan N, Walsh CA, Berezovskii VK, Livingstone MS, Greenberg ME (2016) Evolution of Osteocrin as an activity-regulated factor in the primate brain, Nature, 539, p 242-247.
  2. Greenberg ME, Ziff EB (1984) Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene, Nature, 311(5985), p. 433-438.
  3. Spiegel I, Mardinly AR, Gabel HW, Bazinet JE, couch CH, Tzeng CP, Harmin DA, Greenberg ME (2014) Npas4 regulates excitatory-inhibitory balance within neural circuits through cell-type-specific gene program, Cell, 157(5), p. 1216-1229.
  4. Mardinly AR, Spiegel I, Patrizi A, Centofante E, Bazinet JE, Tzeng CP, Mandel-Brehm C, Harmin DA, Adesnik H, Fagiolini M, Greenberg ME (2016) Sensory experience regulates cortical inhibition by inducing IGF1 in VIP neurons, Nature, 531, p. 371-375.

 

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