Hyaluronan regulates synapse formation and function in developing neural networks

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.

CB1 antagonism increases excitatory synaptogenesis in a cortical spheroid model of fetal brain development

“…This indicates that CB 1 is closer to the presynaptic marker than the postsynaptic marker and preferentially localizes to the presynapse. The median distance between VGLUT-1 and PSD-95 (0.131 µm) was similar to distance between CB 1 and PSD-95 and is consistent with our previous STORM measurements of synaptic cleft size 26 , 27 . Additionally, we investigated CB 1 localization at inhibitory synapses (n = 47) and observed a significantly shorter ( p = 0.014) median distance between CB 1 /presynaptic marker VGAT (0.046 µm) compared to CB 1 /postsynaptic marker gephyrin (0.062 µm) (Fig.…”

“…Disruptions to the spatial and temporal regulation of synaptogenesis during this critical window is thought to drive developmental disorders such as ASD 20 , 55 . At 90 days of development, we have previously demonstrated that our cortical spheroids exhibit both excitatory and inhibitory synapses 26 , 27 . Furthermore, these synaptic connections exhibit a high level of plasticity, and are readily altered by acute perturbations to either the intracellular cytoskeleton or extracellular matrix 26 , 27 .…”

“…At 90 days of development, we have previously demonstrated that our cortical spheroids exhibit both excitatory and inhibitory synapses 26 , 27 . Furthermore, these synaptic connections exhibit a high level of plasticity, and are readily altered by acute perturbations to either the intracellular cytoskeleton or extracellular matrix 26 , 27 . Thus, we have established a window to selectively observe how CB 1 signaling contributes to the initial formation of synaptic connections and subsequent development of synaptic activity.…”

“…This experiment was repeated 3 times with independently grown sets of cortical spheroids. A treatment period of 24 h was chosen to selectively perturb synaptogenesis (rather than migration or differentiation) and prior research from our lab has shown that this is an effective period of treatment for altering synaptogenesis 27 .…”

“…These models, which replicate brain tissue structure better than two-dimensional cell culture 21 , 22 , provide an avenue for drug testing in a genetically relevant paradigm 23 , 24 and allow for the study of human disease processes without complicated in vivo work 20 , 22 . Using IPSCs from neurotypical control patients, we are able to grow cortical spheroids which have functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization which resembles the dorsal telencephalon of the human fetus at 19–24 weeks post conception 25 – 27 .…”