Abstract
Neurodevelopmental disorders present with synaptic alterations that disrupt the balance between excitatory and inhibitory signaling. For example, hyperexcitability of cortical neurons is associated with both epilepsy and autism spectrum disorders. However, the mechanisms that initially establish the balance between excitatory and inhibitory signaling in brain development are not well understood. Here, we sought to determine how the extracellular matrix directs synapse formation and regulates synaptic function in a model of human cortical brain development. The extracellular matrix, making up twenty percent of brain volume, is largely comprised of hyaluronan. Hyaluronan acts as both a scaffold of the extracellular matrix and a space-filling molecule. Hyaluronan is present from the onset of brain development, beginning with neural crest cell migration. Through acute perturbation of hyaluronan levels during synaptogenesis, we sought to determine how hyaluronan impacts the ratio of excitatory to inhibitory synapse formation and the resulting neural activity. We used 3-D cortical spheroids derived from human induced pluripotent stem cells to replicate this neurodevelopmental window. Our results demonstrate that hyaluronan preferentially surrounds nascent excitatory synapses. Removal of hyaluronan increases the expression of excitatory synapse markers and results in a corresponding increase in the formation of excitatory synapses, while also decreasing inhibitory synapse formation. This increased excitatory synapse formation elevates network activity, as demonstrated by microelectrode array analysis. In contrast, the addition of purified hyaluronan suppresses excitatory synapse formation. These results establish that the hyaluronan extracellular matrix surrounds developing excitatory synapses, where it critically regulates synapse formation and the resulting balance between excitatory to inhibitory signaling.
Highlights
It is bound by lectin domains of proteoglycans, such as neurocan, brevican, aggrecan and versican, which are known as lecticans[8]
Neurotypic control skin fibroblasts of the cell line 7545 19B were reprogrammed into human-induced pluripotent stem cells in the laboratory of Dr Mike McConnell (UVa) with the addition of Yamanaka transcription factors Oct3/4, Sox[2], Klf[4], and c-Myc using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Invitrogen). 7545 fibroblasts were obtained under an MTA with the Coriell Institute. 9319 and BOH1 hIPSCs were obtained under a material transfer agreement (MTA) with Kristen Brennand and the Salk Institute
In order to detect the presence of HA machinery, we immunostained 3-month-old cortical spheroid cryosections for the predominant HAS isoform of the cortex, Hyaluronan Synthase 2 (HAS2), as well as the primary HA receptor, CD44
Summary
It is bound by lectin domains of proteoglycans, such as neurocan, brevican, aggrecan and versican, which are known as lecticans[8]. Radial glia project from these regions, allowing for the migration of developing neurons to the cortical plate, a region corresponding to the cortical layers[40] These human models offer an unprecedented research model to identify molecular mechanisms that regulate the development of neuronal activity and alterations that result in diseaseassociated hyperexcitability signatures[39]. We establish a human brain spheroid model of the HA-based ECM and use this model to investigate how HA alterations disrupt neural circuitry at the level of synapse formation and function. The following research tests whether the hyaluronan-based extracellular matrix directs excitatory synapse formation and regulates the resulting neuronal activity. Our data from the onset of the synaptogenesis demonstrates that HA is present and enriched at nascent excitatory synapses as compared to inhibitory synapses At these developing excitatory synapses, HA antagonizes excitatory synapse formation and suppresses the corresponding neural activity. These findings establish HA as a crucial regulator of synapse formation and function
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call