Using in vivo time lapse structural and functional imaging studies, as well as electrophysiology, we are discovering novel cellular and circuit-based mechanisms governing visual system development. We demonstrated that even a brief period of enhanced visual system activity facilitates the development of visual system synaptic connections and circuit function, and we have identified molecular and cellular mechanisms underlying to role of visual experience in circuit development. For instance, repeated exposure to anterior-to-posterior moving stimuli facilitates development of topographic retinotectal maps by an NMDA receptor-dependent mechanism, whereas exposure to posterior-to-anterior moving stimuli interferes with topographic map development.

To discover new players in plasticity, we conducted an unbiased quantitative tandem mass spectrometry-based screen to identify changes in newly synthesized proteins induced by 4h of visual stimulation in intact Xenopus. We used BONCAT, BioOrthogonal Noncanonical Amino Acid Tagging, a metabolic protein labeling strategy in which non-canonical amino acids are incorporated into newly synthesized proteins, which are then tagged with biotin using click chemistry. We were the first lab to conduct quantitative BONCAT experiments in vivo in a vertebrate to identify changes in protein synthesis in response to sensory experience. This strategy led to the identification of unexpected experience-dependent mechanisms that contribute to brain development and plasticity. The finding that visual stimulation regimes promote the development and function of the Xenopus visual system spurred our interest in exploring the potential role of visual experience in promoting recovery from local injury in the visual circuit. These studies have shown that repeated exposure to brief periods of visual experience rehabilitate the injured visual circuit using cellular mechanisms akin to those employed by the developing circuit, as schematized above.

The brain processes information, makes decisions, mediates cognitive and motor outputs through the functions of proteins in different types of connected neurons organized in complex arrangements. Neurons in brain circuits are often intermingled with non-neuronal cells that are also essential to brain function. Dissecting the biochemical processes in different cellular components of neuronal circuits in intact animals is crucial to understand the biology of brain cells and circuits. Because proteins carry out core functions in all cells, and because protein distribution in morphologically complex cells like neurons and glia can influence their function, understanding neuronal and circuit function requires a comprehensive knowledge of proteins underlying the molecular architectures of neurons and their connections, and how the expression and subcellular distribution of neuronal proteins change with brain function.

Current estimates suggest that there are 21,000 genes and between 250,000 and 1 million different proteins in humans. Transcriptional dynamics do not directly correlate with protein dynamics because protein synthesis, turnover and subcellular localization are tightly regulated spatially and temporally after transcription. Most neuroscientists acknowledge that current information about transcriptional dynamics in the brain has been transformative for understanding brain functions and pathologies. Comparable information about spatial and temporal proteomic dynamics, particularly when combined with anatomic and genetic information about the neurons and circuits under study, would be similarly or even more transformative for our understanding of the brain.

Consequently, we launched a new direction of research in the lab to understand brain plasticity and function by studying protein dynamics in the intact brain in response to activity, sensory input, development and aging. We take advantage of state of the art quantitative mass spectrometry methods and protein labeling strategies including bioorthogonal non-canonical amino acid tagging (BONCAT), to investigate in vivo proteomic dynamics with cell type resolution. We expect that identification of proteins whose synthesis and subcellular distribution are altered by activity, sensory experience or over development and aging will serve as an essential first step in probing the function of these brain proteins in plasticity, development or aging, or in formulating hypotheses concerning the molecular mechanisms regulating activity-dependent synaptic dynamics, cortical neuron connectivity, protein distribution and proteostasis in morphologically complex cells like neurons.

Exosomes, a type of EV, are produced by every cell type in the brain and they contain selectively-packaged cargoes such as bioactive lipids, proteins, and diverse RNAs. Thus, exosomes have the capacity to signal to recipient cells and mediate physiological changes, without requiring direct contact between cells. Two different types of studies, one in glioma and the other investigating the development of neuromuscular junction in Drosophila, inspired us to investigate the potential role of exosomes in brain development and function, as well as neurological diseases.

Previous work from our lab used hiPSC-derived neurons as a source of neuronal exosomes to conduct proteomic analysis of exosomes released from hiPSC-derived neurons and isogenic neurons lacking MECP2 as a model of the neurodevelopmental disorder Rett syndrome. Through this study, we found that exosomes from developing neuronal cultures increase proliferation and synaptic density and revert the phenotype caused by the lack of MECP2 (Sharma et al., 2019). Currently, we are establishing a human iPSC-based platform on which to further investigate how exosomes from different neural cell-types and different Alzheimer’s risk genotypes regulate neuronal function and synaptic plasticity.