Neuron-Specific Endosomal Regulators of Excitatory Synaptic Neurotransmission and Synapse Stability

Material below summarizes the article Neuron-Specific Gene 2 (NSG2) Encodes an AMPA Receptor Interacting Protein That Modulates Excitatory Neurotransmission, published on January 4, 2019, in eNeuro and authored by Praveen Chander, Matthew J. Kennedy, Bettina Winckler, and Jason P. Weick.

Normal brain function depends on the proper communication of information among billions of neurons. This communication occurs at specialized junctions called synapses, and each neuron contains approximately a thousand synapses, making the total number of synapses in the human brain on the order of 100 trillion.

Adding to this complexity is the fact that each synapse contains a large number of proteins that function as receptors as well as scaffolding, signaling, and trafficking proteins. While this complexity is beginning to be unraveled, a major outstanding question in the field concerns how synapses are assembled during development, when synapses serving all of these functions form within a short time period. Specifically, we were interested in unraveling how nascent synapses (which contain relatively few proteins) recruit the synaptic protein complement during development.

To address this question, we used neural differentiation of human pluripotent stem cells (hPSCs), which allowed us to observe some of the earliest events during synapse formation. hPSC-derived neurons (hPSNs) establish synapses over a protracted time-course, which permits researchers to ask detailed questions about the timing of specific events.

We used global gene expression analysis of hPSNs to identify several genes with putative roles in synaptogenesis that remained uncharacterized. Among the genes that were significantly upregulated during synaptogenesis we identified neuron-specific gene 2 (NSG2), which was the seventh most upregulated gene among thousands.

NSG2 belongs to the “neuron-specific gene” family (NSG1–NSG3). Interestingly, the NSG family evolved uniquely in vertebrates, with flies, worms, and other invertebrates lacking these proteins. Compared to other cells, neurons have some of the most complex architectures, presenting a unique challenge for trafficking proteins to the correct location in space and time.

For example, proteins destined for synapses are typically synthesized in the cell body and have to be transported to distal locations within multiple dendrites. Because of this morphologic and functional complexity, neurons have evolved specialized vesicular trafficking proteins like those of the NSG family to address this complexity. Previous research has established important roles of NSG1 and NSG3 in diverse synaptic functions, but the function of NSG2 has remained elusive.

We first confirmed NSG2 was present only in neurons and specifically in the soma and in discrete locations (puncta) in neuronal dendrites, in developing hPSNs and cultures taken from developing mouse brains. Heightened expression of NSG2 was found in rodent brain early during development and overlapping with periods of synaptogenesis.

While we predicted these findings, we were surprised a significant subset of NSG2 localized to approximately 40 percent of excitatory synapses. The vast majority of excitatory neurons in the brain respond to the neurotransmitter glutamate via post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs). We found NSG2 localized to areas of neuronal dendrites that contained significant amounts of AMPARs that were on the surface of the cell. This meant NSG2 was likely present at synapses where AMPARs are functional, and was receiving glutamate from presynaptic neurons. Using antibodies specific to NSG2 and AMPARs, we were able to show these two proteins physically interact in the brain and in cultured neurons.

Because of this finding and the fact that previous studies established roles for NSG1 and NSG3 in AMPAR trafficking, we wondered if NSG2 may have a similar or unique role promoting synaptic transmission via AMPAR trafficking. We found eliminating expression of endogenous NSG2 using CRISPR/Cas9 knockout, or increasing NSG2 levels by overexpression, caused alterations in functional neurotransmission. In the case of NSG2 knockout, the resulting functional alterations appeared to be disproportionate to the percent of synapses that contained NSG2. In other words, while NSG2 was only present at 40 percent of synapses, its knockout caused a 60 percent reduction in synaptic transmission.

Most intriguingly, however, these alterations in function were not correlated with predictable or significant changes in AMPAR surface expression, suggesting a novel role of NSG2 in promoting synaptic function that may be independent of bulk AMPAR trafficking. Thus, our findings demonstrate a role for NSG2 in the regulation of AMPAR surface expression and implicate NSG2 as a potential partner of AMPARs in regulated endosomal trafficking in developing dendrites.

How NSG2 modulates excitatory synaptic function specifically remains unclear. One mechanism likely involves secretion of AMPARs via post-Golgi endosomal vesicles that may contain individual or combinations of NSG family members depending on function. Future investigations will aim to answer these questions.

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Neuron-Specific Gene 2 (NSG2) Encodes an AMPA Receptor Interacting Protein That Modulates Excitatory Neurotransmission. Praveen Chander, Matthew J. Kennedy, Bettina Winckler, and Jason P. Weick. eNeuro Jan 2019, 6 (1) 0292–18.2018; DOI: 10.1523/ENEURO.0292-18.2018

Praveen Chander
Praveen Chander is a graduate student in the Weick Lab at the University of New Mexico (UNM) School of Medicine. He earned his bachelor’s degree in India, majoring in microbiology and biotechnology, and his master’s degree at Cleveland State University, where he carried out his thesis research in cancer biology. After working in industry to produce therapeutic monoclonal antibodies, he returned to academia to pursue a PhD in neuroscience, joining the department of neurosciences at the UNM School of Medicine. As a doctoral student in his final year, he has significantly contributed to the molecular and functional characterization of NSG2. He has a strong interest in studying the cellular and molecular aspects of neurodevelopment and its disorders and hopes to be involved in future research that could translate into effective therapeutics.
Jason P. Weick, PhD
Jason P. Weick is an assistant professor in the department of neurosciences at the University of New Mexico (UNM) School of Medicine, where research in the Weick Lab centers on understanding multiple aspects of the development of functional neural circuits. Using neurons differentiated from pluripotent stem cells, the lab studies development under normal conditions and in the context of developmental disorders. It recently identified NSG2, an uncharacterized protein strongly upregulated as neuroepithelial cells differentiate to post-mitotic neurons, but subsequently downregulated in adults. The lab also identified an abnormal pattern of activity in in vitro cultures of neurons that carry trisomy 21, the underlying cause of Down Syndrome. Current investigations seek to understand the molecular cause of this abnormality, as well as the effects of ethanol on developing neurons during early stages of synapse formation.
Praveen Chander
Praveen Chander is a graduate student at the University of New Mexico School of Medicine.

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