How do brain circuits form? What instructs self-assembly of connectivity during development, and how is input from the environment used later in life? How does circuitry emerge from cell biology, and what goes wrong in mental illness and pathologies of brain wiring? Might we be able to ameliorate miswiring in patients?
To get at these questions, our team develops synthetic biology tools and applies them to the developing brain of mice and rats. A big focus is developing new CRISPR tools able to knock in precise genome edits directly in the developing bran, often by in utero electroporation, to track and manipulate neural circuits and their molecules as the brain develops. With these approaches, are investigating the wiring patterns that underlie conditions on the neurodevelopmental spectrum, including, epilepsy, autism, and schizophrenia.
The goal of our group is to understand the in vivo mechanisms that determine circuit formation in development and circuit remodeling in adulthood; to discover how these processes deviate to alter brain circuits in neurodevelopmental conditions and mental illness; and to develop the knowhow and methodologies that may allow therapeutic intervention for the regeneration of circuits lost to disease or trauma.
Let’s get into some specifics:
We recently developed an approach that reveals the local protein and RNA networks within axons that are in the process of forming a connection in the developing brain (Poulopoulos et al, Nature, 2019). The approach combines in utero electroporation, subcellular fractionation, and small-particle fluorescence sorting.
Using RNA-seq and mass spectrometry, we know which molecules concentrate in axons of a particular connection, giving us a “parts list” of the machinery that is implementing connectivity in vivo. Some of the biological processes we are currently following are cell adhesion, mTOR signaling, and local translation.
With this knowledge, we are targeting genes that produce these molecules using in vivo CRISPR genome editing to produce knockout and also knockins. This allows us to not only perform area-specific loss-of-function experiments, but also to observe and track the dynamics of these molecules under endogenous expression and sparse labeling in vivo. With some added fancy footwork in the realm of synthetic biology, we can also functionally hijack the these molecules locally using light as they go through the steps of forming or modifying brain circuitry!
Beyond the formation of long-range connectivity, we look up close at the critical moment when a synapse develops to complete the connection between two neurons. We have a long-term interest in the molecules that organize and coordinate the interactions across the synapse (Poulopoulos et al, Neuron, 2009; Altas et al, BioRxiv, 2022), and with our collaborators in the Blanpied lab across the street -the gurus of synapse nanostructure- we use in vivo CRISPR to tag endogenous synaptic proteins and examine their structure and function in the pre- and postsynaptic apparatuses.
Mental illness and pathologies of brain wiring
We use in utero electroporation, in vivo genome editing, and 3D imaging to uncover the effects on brain circuitry of genetic variants associated with neurodevelopmental disorders and Mental illness. By introducing these variants using in vivo CRISPR in select circuits of the rodent brain, we aim to identify how these connections are altered to discover circuit underpinnings of conditions such as epilepsy, autism, and schizophrenia.
For these clinically relevant projects, we work closely with collaborating labs on campus. On our epilepsy projects we work with the labs of Scott Thompson from the department of Physiology, and Peter Crino from the department of Neurology to create a full workflow from molecular, cellular, and electrophysiological endophenotyping, to EEG and behavioral monitoring in order to analyze clinically relevant mutations in mice .
On Schizophrenia, we collaborate with groups at the Maryland Psychiatric Research Center and Seth Ament at the Institute for Genome Sciences, who provide invaluable human brain imaging and genome data from cohorts of patients, which we get to test in the mouse cerebral cortex. On neurodevelopmental disorders we collaborate with the groups of Saima Riazuddin and Zubair Ahmed who have used human genetics of isolated populations to discovered a host of new recessive gene mutations leading to sever disorders of brain development, which we can examine molecularly in the embryonic mouse brain.
To go beyond descriptions of circuit aberrations, we have developed ESI, the Endophenotype Severity Index, a ratiometric value that allows generalizable quantification and modeling of circuit alterations associated with disease. With an interest in the circuitry of the cerebral cortex, with ESI we hope to document specific circuit and network defects in mouse models of mental illness, such as schizophrenia. Working together with our neighbors and collaborators in the Mathur lab, we can examine both connectivity and function when looking at what circuit defects underly mental illness.
Optimizing in vivo knockin with Cas9-RC (did we mention, in vivo?)
We are in constant need of better tools. For instance, we would like a better way to edit the genome directly in relevant cells in situ, both for experimental purposes, and for future therapeutics. We’ve been tinkering with Cas9, using variants, mutants, and chimeras, as well as new delivery methods in vivo. All this so we can develop a way of manipulating the genome of desired cells with specificity, with minimal off-target effects or adverse reactions, and with high yield. Check out Richardson et al. CRISPR J, 2023.
Hodaptics: cell-cell interactions in intact systems
The biology of cell-cell interactions is a beautifully complex and critical part of the development and function of multicellular systems. In the nervous system, this biology manifests in its extreme, with a given neuron directly contacting a bewildering and perpetually-changing number of direct cellular neighbors at any given time. It is this multitude and dynamics of cell-cell interactions that give nervous tissue its unique functionalities. However, the biology of cell-cell interactions is particularly difficult to study, as it requires experimenting on intact systems, while being able to detect and influence elusive transient interactions. To allow experimentation into this interesting biology, we have developed and are currently optimizing a new genetic technology, termed “hodaptics” (from Greek hodos- road, and haptein -to touch), that will allow us to detect and manipulate transient cell-cell interactions as they occur within intact systems such as the developing and learning brain, as well as other complex multicellular systems.