Many brain disorders exhibit altered synapse formation in development or synapse loss with age. To understand the complexities of human synapse development and degeneration, scientists now engineer neurons and brain organoids from human-induced pluripotent stem cells (hIPSC). These hIPSC-derived brain models develop both excitatory and inhibitory synapses and functional synaptic activity. In this review, we address the ability of hIPSC-derived brain models to recapitulate synapse development and insights gained into the molecular mechanisms underlying synaptic alterations in neuronal disorders. We also discuss the potential for more accurate human brain models to advance our understanding of synapse development, degeneration, and therapeutic responses.

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.

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Synapses are specialized neural cell adhesions that underlie neurotransmission (Lynch, Rex, & Gall, 2007; Vicente-Manzanares, Hodges, & Horwitz, 2009). Synapse development is altered in the neurodevelopmental disorders of epilepsy, intellectual disability, and autism spectrum disorders (Forrest, Parnell, & Penzes, 2018). The onset of excitatory synapse formation occurs during mid-fetal gestation (Tierney & Nelson, 2009; Yuste & Bonhoeffer, 2004). Intriguingly, the expression profile of genes associated with neurodevelopmental disorders peaks in prenatal brain development (Birnbaum, Jaffe, Hyde, Kleinman, & Weinberger, 2014). However, due to our inability to image synaptogenesis in utero, prenatal brain development remains understudied. Instead, most research focuses on postnatal refinement of synaptic circuits. In order to research the molecular mechanisms of fetal synaptogenesis, we develop human cortical spheroids (HCSs). HCSs offer an unprecedented opportunity to observe the initial formation of excitatory synapses (Wilson & Newell-Litwa, 2018).

The hyaluronan-based extracellular matrix is expressed throughout nervous system development and is well-known for the formation of perineuronal nets around inhibitory interneurons. Since perineuronal nets form postnatally, the role of hyaluronan in the initial formation of neural circuits remains unclear. Neural circuits emerge from the coordinated electrochemical signaling of excitatory and inhibitory synapses. Hyaluronan localizes to the synaptic cleft of developing excitatory synapses in both human cortical spheroids and the neonatal mouse brain and is diminished in the adult mouse brain. Given this developmental-specific synaptic localization, we sought to determine the mechanisms that regulate hyaluronan synthesis and signaling during synapse formation. We demonstrate that hyaluronan synthase-2, HAS2, is sufficient to increase hyaluronan levels in developing neural circuits of human cortical spheroids. This increased hyaluronan production reduces excitatory synaptogenesis, promotes inhibitory synaptogenesis, and suppresses action potential formation. The hyaluronan receptor, CD44, promotes hyaluronan retention and suppresses excitatory synaptogenesis through regulation of RhoGTPase signaling. Our results reveal mechanisms of hyaluronan synthesis, retention, and signaling in developing neural circuits, shedding light on how disease-associated hyaluronan alterations can contribute to synaptic defects.