Hyaluronan regulates synapse formation and function in developing neural networks

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.

2D differentiated monolayer culture. Neural Progenitor Cells (NPC) were generated from embryoid bodies according to Brennand et al. 2011. NPCs were differentiated or maintained for one week before analysis. NPCs were plated at 50,000 cells per well onto 12 well plates (Corning), precoated with polyornithine (Sigma Aldrich) and laminin (Corning), in NPC media: DMEM/F-12 + GlutaMAX, 1% N-2 Supplement, and 2% B27 without vitamin A (Gibco), 1 μg/mL laminin (Corning) and 20 ng/mL basic fibroblast growth factor (bFGF) (PeproTech). After 24 h NPCs were either differentiated toward neurons (neural differentiation media) or astrocytes (astrocyte differentiation media), or maintained in NPC media. Media was changed every other day for one week before analysis.
Neural was dissolved in neurobasal medium for 24 h before use. Hyaluronan was added to culture medium at 250ug/ mL. Streptomyces Hyaluronidase (Sigma Aldrich 37259-53-3) was added directly to culture medium at 10 U/ mL. Treatment lasted 24 h before harvesting for analysis or fixing for immunohistochemistry. Adenoviral constructs were used for the overexpression of HAS2 in cortical spheroids (Fig. S3). The constructs used were prepared in the same manner as Ishizuka et al. 42 . immunohistochemistry. Cortical spheroids were fixed in 4% paraformaldehyde for 24 h and placed in 30% sucrose for 24 h. Spheroids were then embedded in OCT mounting media overnight (Sakura Finetek USA), flash frozen, and cryosectioned into 10 μm thick sections. Cryosections were permeabilized with 0.2% Tri-tonX-100 in 1 × PBS before immunostaining. Primary antibodies were diluted in 2% normal goat serum in PBS, added to fixed cultures and kept at 4 °C overnight. After three PBS washes secondary antibodies diluted in 2% normal goat serum in PBS were added to fixed cultures and kept at room temperature for 1 h. Cryosections were mounted using Fluoro-gel II with DAPI mounting medium (Electron Microscopy Sciences) for confocal imaging or Vectashield without DAPI (Vector Laboratories 101098-042) for STORM imaging.
2D cultures were fixed after 1 week in 4% paraformaldehyde, 4% sucrose, 1 × PBS. Cells were permeabilized with 0.2% TritonX-100 in 1 × PBS before staining. Primary antibodies were diluted in 2% normal goat serum in PBS, added to fixed cultures and kept at 4 °C overnight. After three PBS washes secondary antibodies diluted in 2% normal goat serum in PBS were added to fixed cultures and kept at room temperature for 1 h. Cultures were mounted using Fluoro-gel II with DAPI mounting medium (Electron Microscopy Sciences).
HA was visualized using Hyaluronic Acid Binding Protein (HABP). HABP is a biotinylated link protein G1 domain of the proteoglycan versican, which binds selectively to HA. Fluorescently labeled streptavidin was used to identify HABP. See supplemental tables 1 and 2 (in the Supplementary tables) for further information. After immunostaining, cryosections were subsequently imaged using a ZEISS LSM 700 confocal or Nikon Ti2-E inverted STORM microscope. All immunohistochemistry was performed in triplicate on at least three different sets of spheroids. confocal microcopy. Cortical spheroid sections were imaged on a Zeiss LSM 700 confocal microscope at 40 × total magnification, using the 639, 555, 488 and 405 channels. Z-stacked images were acquired, 5 images across 5 μm, and merged in ImageJ to generate maximum intensity projections. Confocal images were further analyzed using ImageJ 1.52a analysis software (https ://image j.nih.gov/ij/). Each 4-channel image was analyzed as an 8-bit tiff-file. All samples of the same experiment had equal threshold values for each channel. 10 area samples measuring 100 μm in diameter were used to determine the intensity and density of the fluorescent markers along the edge of the cortical spheroid, coinciding with the cortical plate location. Spheroids were stained with pre-and post-synaptic markers. To analyze the total synapse area, we used the ImageJ colocalization plug in. This produced an image indicating areas where thresholded pre-and post-synaptic markers colocalized by intensity ratio of 10% or more. The area of each individual marker as well as the colocalized synapse area was measured and normalized to DAPI. Normalization to nuclei density (DAPI) has been performed in other brain organoid studies to compare regions of similar cell densities 43,44 , and to evaluate synaptic changes 45 The raw images  are collected with the 3D STORM lens to a back-thinned Princeton Instruments Pro-EM-HS EMCCD 512 × 512 camera and acquired and analyzed with NIS-Elements STORM modules. Processed .nd2 files were analyzed in ImageJ. .nd2 files were opened as a hyperstack, 3D projection was then used to plot the brightest point with the Y-axis as the axis of rotation. A Gaussian blur filter with a radius of 2.00 pixels was applied to avoid bias. A 10 pixel line width was used to analyze of the intensity of synaptic molecules using the plot profile function of ImageJ. Please note that the intensity profile in Fig. 2  RNA concentration was quantified using a NanoDrop One spectrophotometer. 50-100 ng of total RNA was hybridized with reporter and capture probes for nCounter Gene Expression code sets (Neuropathology Codeset) according to the manufacturer's instructions (NanoString Technologies). Using the nSolver analysis system data were normalized to spiked positive controls and housekeeping genes. Transcript counts less than the mean of the negative control transcripts plus 2std for each sample were considered background. Nanostring mRNA analysis was performed on RNA harvested from three sets of cortical spheroids.

Results
characterization and manipulation of hyaluronan in a human cortical spheroid model. We first established that human cortical spheroids contain the proper machinery for HA synthesis and signaling, and that they do, in fact, synthesize an HA-based ECM at the time of synaptogenesis. Consistent with others, we have previously established that discrete synapses are present after 3 months of brain spheroid culture 39 . 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, HAS2, as well as the primary HA receptor, CD44. As shown in Fig. 1A, cortical spheroids endogenously express HA (detected using HABP), HA synthase, HAS2, and HA receptor, CD44 (Fig. 1A,B). Furthermore, HA machinery is enriched within the cortical plate surrounding the ventricles. As demonstrated in Fig. 1C, this region is enriched for neurons and serves as the site of synaptogenesis. In this region, HA is associated with both neurons and astrocytes ( Fig. 1C,D, Fig. S2).
Hyaluronan is present at nascent excitatory synapses. After establishing that cortical spheroids express an HA-based matrix, we next sought to determine whether HA is present at developing synapses ( Fig. 2A). Using confocal imaging, we observed that HA is preferentially enriched at excitatory synapses ( Fig. 2B,C, 4). To further establish where HA is present at nascent excitatory synapses, we used super-resolution STochastic Optical Reconstruction Microscopy to resolve individual excitatory synapses. The pre-and postsynaptic compartments are separated by a synaptic cleft that is approximately 20 nm wide, as measured by electron microscopy 9 . Since confocal microscopy is limited to a resolution of ~ 200 nm, it is unable to distinguish between pre-and post-synaptic compartments 47 . By contrast, STORM offers an approximately tenfold increase in image resolution, with a resolution limit of ~ 20 nm 46 . Thus, STORM microscopy enables detection of distinct pre-and post-synaptic compartments. Using STORM, we identified pre-synaptic compartments by the presence of the vesicular glutamate transporter-1 (vGlut1), the predominant glutamate transporter in our HCSs. To identify adjacent post-synaptic compartments, we immunostained for the scaffolding protein, Post-Synaptic Density-95 (PSD-95). After fluorescent labeling of excitatory synapses and HA, we used three-color STORM, which revealed that HA is present at nascent excitatory synapses. In the majority of cases, HA is sandwiched in the synaptic cleft directly between the pre-and post-synaptic compartments ( Fig. 2D-F). Measurement of the peak Scientific RepoRtS | (2020) 10:16459 | https://doi.org/10.1038/s41598-020-73177-y www.nature.com/scientificreports/ intensity for the presynaptic vGlut-1, HA, and post-synaptic PSD-95 puncta across multiple (167) synapses demonstrated that HA is positioned equidistant from the pre-and post-synaptic compartments (Fig. 2E). Distance from the pre-to post-synaptic marker ( Fig. 2G) is about 100 nm, consistent with our previous observations 48 . In contrast to studies of mature mammalian synapses, in which HA surrounds the synapse but is absent from the synaptic cleft 9,49 , this result suggests that during synapse formation, HA is uniquely positioned between pre-and post-synaptic compartments. Given this close association with pre-and post-synaptic compartments of excitatory synapses, the following experiments address whether HA regulates synaptogenesis and the corresponding emergence of spontaneous synaptic activity.
Hyaluronan manipulations alter mRnA expression of synapse markers. After establishing that cortical spheroids express an HA-based matrix, we sought to acutely perturb HA levels during synapse formation. In order to manipulate endogenous HA levels, we used the following experimental protocols as detailed in Fig. 3A. Day 90 spheroids reflect fetal neurodevelopment of the dorsal forebrain around 18 weeks post-conception, corresponding with the onset on synaptogenesis 39 . In order to acutely increase HA levels, day 90 spheroids were treated with purified HA. Conversely, in order to decrease HA levels, day 90 spheroids were treated with streptomyces hyaluronidase to digest HA. After 24 h of treatment, spheroids were processed for subsequent analysis. We used HABP staining to confirm that we had successfully manipulated HA levels (Fig. S1). Notably, hyaluronidase significantly decreased HA levels, whereas purified HA incorporated into the spheroids and significantly increased HA levels (Fig. S1), thus demonstrating that we are able to acutely manipulate the HA levels of our system. To initially examine the neural pathways impacted by acute hyaluronan perturbations in an unbiased fashion, we employed Nanostring nCounter platform to analyze transcript expression changes in over 700 mRNA targets associated with neuropathologies. mRNA was harvested from 90-day-old cortical spheroids and was subjected to Nanostring nCounter mRNA analysis using the Neuropathology panel. Unlike other targeted transcript analyses, Nanostring directly measures mRNA levels, and does not require intermediate cDNA synthesis. This increases the sensitivity of the Nanostring nCounter platform for low-abundance transcripts, while also preventing the introduction of artifacts through cDNA synthesis 50 . We compared pathway scores between groups of genes known to be involved in neuropathology (Fig. 3B). Pathway scores were plotted for + HA (hyaluronan addition) and − HA www.nature.com/scientificreports/ (hyaluronidase treatment), revealing three pathways that were preferentially increased in response to hyaluronidase treatment: Transcription and Splicing, Growth Factor Signaling, and Neuronal Cytoskeleton (Fig. 3B). This suggests that HA manipulation is sufficient to elicit changes in neuronal physiology. We then compared individual transcript expression levels to further determine the effects of HA manipulation. To visualize the differential expression of individual mRNA transcripts, we plotted transcript level fold change between hyaluronan removal versus hyaluronan addition in a volcano plot (Fig. 3C). Individual transcripts with significantly altered expression in response to hyaluronan manipulation are all associated with synapses, including SNAP91, SLC4A10, SHANK2, and GRIA2, all four of which are upregulated in response to HA degradation (Fig. 3D). At the pre-synaptic compartment, SNAP91 is involved in clathrin coat assembly, neurotransmitter secretion, and synaptic vesicle budding from the pre-synaptic endocytic zone membrane 51 . Within the excitatory post-synaptic compartment, SHANK2 is a post-synaptic scaffolding protein and GRIA2 is glutamate ionotropic receptor AMPA type subunit, both of which mediate excitatory neurotransmission 52,53 . Also in the post-synaptic compartment, SLC4A10 is associated with sodium channels in principal and inhibitory neurons 54 . These data demonstrate that acute HA manipulation for 24h has immediate and detectable effects on synapse-associated transcripts.
The following aims address whether these synaptic mRNA transcript changes correspond with altered synapse formation and function.
Acute HA manipulation alters excitatory synapse formation. To assess whether HA manipulations alter the formation of excitatory synapses, we similarly treated 90-day-old cortical spheroids with either hyaluronidase to remove hyaluronan or the addition of purified high molecular-weight HA (1.0-1.8 MDa) for 24 h (Fig. 3A). This high molecular weight HA is consistent with endogenous neural HA, which is 1-10 MDa 8 . As noted above, this acute 24h treatment was sufficient to induce significant changes in the HA levels of cortical spheroids, decreasing HA levels in response to hyaluronidase, and increasing HA incorporation into extracellular matrix with the addition of purified HA (Fig. 4A,B, S1). Using immunohistochemistry, we identified excitatory synapses by the co-localization of pre-synaptic vGlut-1 and post-synaptic PSD-95 (Fig. 4). In response to decreased HA, the density of excitatory synapses, as identified by the co-localization of vGlut-1 and PSD-95, increased (Fig. 4A,C). Individually, both vGlut-1 and PSD-95 area increased in response to HA removal, demonstrating that the effects of HA removal are not specific for distinct pre-or post-synaptic compartments (Fig. 4A,B). By contrast, elevating HA levels decreased the area of vGlut-1, PSD-95, and excitatory synapses, which were identified by the co-localization of vGlut-1 and PSD-95 (Fig. 4). These results indicate that HA normally functions to restrict excitatory synapse formation.

Acute HA manipulations result in corresponding changes in inhibitory synapse formation.
To determine whether these effects on excitatory synaptogenesis lead to corresponding changes in inhibitory synapse formation, we immunostained cortical spheroid cryosections for inhibitory synapse markers. We used the vesicular GABA transporter (vGAT) to identify the pre-synaptic terminal of inhibitory synapses, and the scaffolding protein, gephyrin, to identify post-synaptic compartments of inhibitory synapses (Fig. 5A). In contrast to the effects on excitatory synapse formation in response to hyaluronan manipulations (Fig. 4), we observed opposite effects on inhibitory synapse formation (Fig. 5). We stratified all cortical spheroids based on HA levels. This analysis highlighted how inhibitory synapse formation directly scales with HA levels (Fig. 5C). Furthermore, at the level of individual pre-and post-synaptic markers, we observed increases in both pre-synaptic vGAT area and to a greater extent, post-synaptic gephyrin area (Fig. 5B). Thus, HA-mediated suppression of excitatory synapse formation inversely correlates with increased inhibitory synapse formation.

Hyaluronan critically regulates neuronal excitability.
Our results thus far demonstrate that hyaluronan removal promotes excitatory synapse formation and decreases inhibitory synapse formation (Figs. 4, 5).
In an opposite fashion, hyaluronan addition decreases excitatory synapse formation and increases inhibitory synapse formation (Figs. 4, 5). Given the observed changes in synapse formation, we hypothesize that HA func-  www.nature.com/scientificreports/ tions to attenuate neuronal excitability. Neuronal hyperexitability is cytotoxic and contributes to brain disorders such as autism, epilepsy, and intellectual disability 12,27 . In order to assess the effects of hyaluronan manipulation on neuronal activity, we dissociated 76-day-old cortical spheroids onto microelectrode arrays (Fig. 6A,E). After two weeks on microelectrode surfaces, the dissociated cortical spheroids re-establish neuronal connections and their extracellular matrix (Fig. 6B). After this two-week recovery period, we recorded the baseline neural activity, followed by HA manipulation and recording of neural activity for the 24 h (Fig. 6C), the time period in which we observed changes in synapse formation (Figs. 4, 5). Finally, we used tetrodotoxin treatment to suppress action potential formation, verifying that we were recording spontaneous neural activity (Fig. 6D). After 6-12 h of hyaluronidase treatment, HA removal significantly elevates neural activity over untreated and hyaluronan addition. Conversely the addition of HA has an opposing trend towards depressed neural activity, beginning around 24 h (Fig. 6D). Since cortical spheroids express endogenous HA (Figs. 1, 6B, and Fig. S1), we suggest that the addition of HA requires further time to incorporate into the synaptic ECM and destabilize synaptic structures. Thus, we only begin to observe the effects of HA addition at later timepoints. Overall, these results suggest that hyaluronan prevents the emergence of a hyperexcitable state in developing neural networks. Through these protocols, we have successfully established methodology to acutely manipulate HA levels in cortical spheroids and observe the resulting impact on synapse formation (Figs. 3, 4, 5) and function (Fig. 6).

Discussion
Through this research, we demonstrate that HA critically regulates synapse formation and function in early human cortical brain development. We established that human cortical spheroids serve as an ideal model for studying the role of the ECM in neural circuit formation. We confirmed that cortical spheroids are capable of producing their own endogenous ECM and explored this in further detail to determine how HA interacts with www.nature.com/scientificreports/ developing synapses. We found that HA is preferentially present at the synaptic cleft of excitatory synapses compared to inhibitory synapses ( Fig. 2 and S4). These data are in contrast to studies conducted during later brain development, when synapses have matured. At these mature synapses, previous data suggests that HA is not present in the synaptic cleft in adult synapses, but instead surrounds and encapsulates synapses 9,49 . This suggests a temporally important regulatory role for HA at developing synapses. It has been shown that HA is highly increased in the brain during this pre-natal developmental window 55 . Furthermore, several other neurodevelopmental processes leading up to synapse formation, such as neurulation and neural crest cell migration, are dependent upon HA concentration 1,3 . Based on the crucial roles of HA leading up to synaptogenesis and our data highlighting its presence at the developing synapse, we hypothesized that HA critically regulates the formation and function of synapses in developing neural networks. Using human brain spheroids, we were able to address how hyaluronan manipulations directly impact the initial formation of synaptic contacts. Using Nanostring transcriptional analysis, we initially observed increased expression of synaptic mRNAs in response to hyaluronan removal. These synaptic transcripts include SNAP91, SLC4A10, SHANK2, and GRIA2. SNAP91 (also referred to as F1-20) expression has been associated with excitatory synapse maturation 56 , suggesting that hyaluronidase may accelerate excitatory synapse development. Similarly, overexpression of GRIA2, a glutamate receptor subunit is sufficient to induce dendritic spine formation 57 . Furthermore, SLC4A10 promotes neuronal excitability, with slc4a10-/-mice exhibiting reduced epileptic seizure susceptibility 58 , while SHANK2 regulates neuronal excitability through NMDA receptor function 59 . Together, the affected transcripts suggest that hyaluronan removal promotes a hyperexcitable state, which we demonstrated through further analysis of synapse formation and function. HA manipulation alone was sufficient to alter the excitatory to inhibitory synaptic ratio and the resulting synaptic activity (Figs. 3, 4, 5 and 6).
Our results establish a new role for HA during the formation of synapses, that further highlights HA as a critical regulator of synaptic plasticity throughout brain development. For example, HA has previously been described as a regulator of neuroplasticity through perineuronal nets. Depletion of HA through degradation of perineuronal nets in mice revealed increased amplitude of action potentials at the excitatory postsynaptic terminal 60 . However, perineuronal nets form during postnatal week 3-5 in mice 61 , a timepoint corresponding to about 4-11 years in humans 62 . Perineuronal nets stabilize mature synapses on a subset of inhibitory neurons by restricting neurite growth and synapse formation 7,8 . The dense ECM meshwork of perineuronal nets is thought to protect the specified neuron from hyperexcitability. However, HA is also present in the interstitial space between neurons 12 . In our data, we observed HA concentrated in the developing cortical plate of brain spheroids (Fig. 1), where a significant portion localized to nascent excitatory synapses (Fig. 2) and regulated the emerging neural activity (Fig. 6). Consistent with our data that HA can regulate brain physiology independent of the formation of perineuronal nets, HAS3 −/− mice exhibited unperturbed perineuronal nets, yet these mice suffered hippocampal www.nature.com/scientificreports/ seizures 12 . Together with our results, these data further demonstrate that HA can regulate neuronal excitability independent of peri-neuronal nets, by directly affecting synaptic signaling events 16 .
To appreciate the diverse mechanisms by which HA may regulate synapse formation and function, it is necessary to evaluate the physiological and pathophysiological roles of the HA-based ECM in other tissue systems. HA has been most heavily explored in cartilage, where it provides strength, maintains space, and resists shear forces and compression. HA is required for these mechanical functions, which rely on the retention of bound proteoglycans 63 . In cartilage, HA has additionally been shown to regulate chondrocyte maturation 18,64 . In skin keratinocytes, HA-CD44 interactions have been shown to affect proliferation, survival, migration, cell-cell adhesion, and differentiation 65 . Similarly, in metastatic breast cancer, the interaction of HA with the CD44 receptor mediates tumor cell migration 1,2,4 . Translating these diverse roles into brain physiology, it is known that HA is required for cell migration during neurulation and that HA has effects on proliferation and differentiation in the hippocampal sub-granular zone of mice 12,16 . Furthermore, HA deficiency in mouse HAS isoform knockouts prevents extracellular water retention and reduces the size of the extracellular space, similar to the space-filling role of HA in cartilage; this reduced extracellular space elicits epileptic seizures through a postulated increase in neurotransmitter diffusion 12,16 . Notably, restored osmolarity was sufficient to reduce neuronal excitability 12 . Consistent with the space-filling role of HA, our data supports a model whereby HA restricts the available space across all treatment groups, and were used to bin the corresponding areas of inhibitory synapse marker, gephyrin and vGAT. (C) Quantification of the resulting changes total inhibitory synapse area as determined by co-localized pre-and post-synaptic markers normalized to DAPI. Inhibitory synapse area is stratified based on the corresponding HA levels. n = 30 spheroid slices per treatment. Solid black dots represent 95 th percentile outliers, one asterisk signifies p value < 0.05, two asterisks signify p value < 0.01. www.nature.com/scientificreports/ for pre-and post-synaptic membranes to form and contact one another. In this model where HA serves a spacefilling role, one would predict that HA would predominantly suppress the formation of excitatory synapses, which form on post-synaptic actin-rich protrusions, as opposed to inhibitory synapses which form along the dendrite. Multiple lines of evidence suggest that HA preferentially restricts excitatory synapse formation, including the selective enrichment of HA at excitatory synapses (Fig. 2), and HA-mediated reductions in excitatory synapse area (Fig. 4) as contrasted with increased inhibitory synapse formation with increasing HA levels (Fig. 5). Since HA does not restrict, but rather promotes, inhibitory synapse formation, we suggest that these effects may be indirectly driven by competition for shared synaptic proteins. For example, competition for the adhesive neurexinneuroligin complex regulates the balance between excitatory to inhibitory synapse formation and signaling 17 . These inverse changes in synapse formation exacerbate imbalances in the emerging ratio between excitatory and inhibitory signaling, resulting in a hyperexcitable state in response to HA removal (Fig. 6). While most research on the loss of HA in pathological conditions focuses on the loss of physical extracellular space, HA also regulates intercellular signaling through its interaction with its known cell surface receptors, RHAMM and CD44, of which the most abundant is CD44 8 . Both RHAMM and CD44 are expressed by neurons 3 . While RHAMM is expressed in neurons, it predominantly regulates cell migration and cell cycle in astrocytes and microglia, where it has been shown to interact with microtubules and the actin cytoskeleton 66 . The predominant HA receptor, CD44, is found in neurons, astrocytes and neural progenitor cells, and has been visualized in our model (Fig. 1). The signaling mechanism of this transmembrane receptor is not yet fully understood. HA binds to N-terminal motifs of CD44 that act as loading sites, giving rise to clusters as it binds to HA outside the cell 67 . This alone is hypothesized to cause cellular response through changes in force on the cell surface that are balanced by intercellular attachment of CD44 to the actin cytoskeleton through ERM complexes 18,20,65 . CD44 overexpression has been linked to changes in astrocyte morphology. CD44 has also been linked to RhoGTPase actin cytoskeletal regulators, Rac1 and RhoA, in astrocytes as well as other systems such as breast cancer cells and keratinocytes 21,68 . However, a canonical signaling pathway for CD44 has not yet been agreed upon, and CD44 may also signal outside of HA binding. For example, MMP-mediated cleavage of the extracellular domain of CD44 can regulate CD44 signaling pathways 68 . Thus, while many studies have examined the role of ECM receptors, such as CD44, fewer have actually examined the direct impact of ECM alterations on synapses as we have done.

Scientific
Although further work is needed to parse out the mechanism by which HA orchestrates neural circuit formation, we have demonstrated significant synaptic changes at the mRNA, protein, and functional levels in response to HA manipulations. Through this research, we demonstrated a novel role for the HA-ECM in the establishment of the E/I ratio of developing networks. In particular, HA removal was sufficient to drive a hyperexcitable state, which is characteristic of neurodevelopmental disorders, including epilepsy, intellectual disability, and autism spectrum disorders 12,35,40 . Conversely, our observation that HA decreases excitatory synapse formation could have implications for aging and Alzheimer's Disease, both of which exhibit HA accumulation and synapse loss [69][70][71][72] . In particular, aging is associated with loss of synapses that form on highly plastic thin spines 69 , which resemble the filopodia-like spine precursors involved in prenatal synaptogenesis 30,48,73 . Thus, the findings in the study could have relevance for a spectrum of neurologic conditions, from neurodevelopmental disorders to agerelated cognitive decline. In conclusion, our data is the first to demonstrate direct integration of HA between the pre-and post-compartments of developing excitatory synapses. Further modulation of HA and associated ECM components will advance our understanding of the physiological roles of ECM in early brain development and identify potential therapeutic targets for neurological disorders.